Bioresource Technology 89 (2003) 145–154

Comparative biosorption of mercuric ions from aquatic systems

by immobilized live and heat-inactivated Trametes versicolor and
Pleurotus sajur-caju
M.Y. Arıca a, C € . Gencß
ß . Arpa b, B. Kaya a, S. Bektasß b, A. Denizli b, O b,*

a
Department of Biology, Kırıkkale University, 71450 Yahsßihan-Kırıkkale, Turkey
b
Department of Chemistry, Hacettepe University, 06532 Beytepe-Ankara, Turkey
Received 6 November 2001; received in revised form 27 January 2003; accepted 30 January 2003

Abstract
Trametes versicolor and Pleurotus sajur-caju mycelia immobilized in Ca-alginate beads were used for the removal of mercuric ions
from aqueous solutions. The sorption of Hg(II) ions by alginate beads and both immobilized live and heat-killed fungal mycelia of
T. versicolor and P. sajur-caju was studied in the concentration range of 0.150–3.00 mmol dm
3 . The biosorption of Hg(II) increased
as the initial concentration of Hg(II) ions increased in the medium. Maximum biosorption capacities for plain alginate beads were
0.144  0.005 mmol Hg(II)/g; for immobilized live and heat-killed fungal mycelia of T. versicolor were 0.171  0.007 mmol Hg(II)/g
and 0.383  0.012 mmol Hg(II)/g respectively; whereas for live and heat-killed P. sajur-caju, the values were 0.450  0.014 mmol
Hg(II)/g and 0.660  0.019 mmol Hg(II)/g respectively. Biosorption equilibrium was established in about 1 h and the equilibrium
adsorption was well described by Langmuir and Freundlich adsorption isotherms. Between 15 and 45 °C the biosorption capacity
was not affected and maximum adsorption was observed between pH 4.0 and 6.0. The alginate-fungus beads could be regenerated
using 10 mmol dm
3 HCl solution, with up to 97% recovery. The biosorbents were reused in five biosorption–desorption cycles
without a significant loss in biosorption capacity. Heat-killed T. versicolor and P. sajur-caju removed 73% and 81% of the Hg(II)
ions, respectively, from synthetic wastewater samples.
Ó 2003 Elsevier Science Ltd. All rights reserved.

Keywords: Hg(II); Alginate beads; Entrapment; Biosorption; Trametes versicolor; Pleurotus sajur-caju

1. Introduction Several chemical processes have been utilized for

Mercury is one of the most toxic heavy metals and it
has been responsible for most serious outbreaks of metal
poisoning, e.g., Minamata disease (Kurtland et al.,
1960). Many areas in the world are contaminated by
mercury, posing serious environmental problems. Mer-
cury in any form introduced to the natural environment
from a variety of sources is converted into toxic volatile
forms, i.e., methylmercury chloride, ethylmercury chlo-
ride and phenylmercury chloride by micro-organisms
and abiotic processes. Bioaccumulation of alkylmercury
through food chains causes a potential risk to consu-
mers of fish and shellfish (Pak and Bartha, 1998; Spry
and Wien, 1991).
*
Corresponding author. Tel.: +90-312-299-20-19.
E-mail address: omer@hacettepe.edu.tr (O. Gencß).
removal of mercury from mercury-contaminated in-
dustrial wastewater, but it is difficult to apply chemical
processes to purification of mercury contaminated soils
and water because they need enormous amounts of
chemicals, and lead to secondary pollution (Habashi,
1978). Common treatment systems to remove mercury
from contaminated water are mostly based on sorption
to material such as ion exchange resins (Osteen and
Bibler, 1991). In order to develop economic, practical
and efficient techniques, fungal and other microbial
biomass have been used to remove and recover metallic
elements such as mercury (Volesky and Holan, 1995).
Processes for biological mercury removal include mainly
biosorption, bioaccumulation and reduction. Sa glam
et al. (1999) reported biosorption of mercuric ion and
mercuric species by heat inactivated white-rot fungal
cells, Phanerochaete chrysosporium ME 448. Chen and
Wilson (1997) reported bioaccumulation of mercuric ion

0960-8524/03/$ - see front matter Ó 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-8524(03)00042-7
146 M.Y. Arıca et al. / Bioresource Technology 89 (2003) 145–154

by genetically engineered E. coli JM 109 expressing soluble in aqueous solutions and precipitates in the form
metallothionein and mercuric ion transport system. of a coacervate in the presence of multivalent metal ions
Application of fungal biomass to remove heavy like Ca2þ , Co2þ , Fe2þ , Fe3þ and Al3þ . Alginate has been
metals from industrial wastewater and/or to recover previously used as a support material for the immobi-
economically valuable metals is attractive for industry. lization of several enzymes and microbial cells (Jianlog
Many fungal species such as Pseudomonas aeruginosa et al., 2000).
(Chang and Law, 1998), Pycnoporus sanguineus (Zul- In this study, T. versicolor and P. sajur-caju were
fadhyl et al., 2001), Mucor miehei (Fox and Walsh, entrapped using Na-alginate as the natural polymeric
1982), and Aspergillus niger (Aksu et al., 1999) have matrix. After uniform growth on the surface of the
been extensively studied for heavy metal biosorption matrix, the immobilized live and heat-killed fungal
and the process mechanisms seem to be dependent on biomass were used for the biosorption of mercuric ions
species. Relatively few studies have been conducted with from aqueous solutions in a batch system. The effects of
white-rot fungi in detoxifying metal effluents. They are pH, adsorption time, initial metal ion concentration
basidiomycetes which include all the higher fungi that were studied. The effectiveness of desorbing agent (HCl)
are characterized by their sexual fruiting bodies (Okino in stripping adsorbed Hg(II) ions from the alginate
et al., 2000; Guibal et al., 1992; Kapoor et al., 1999). beads and from both immobilized fungal preparations
Trametes versicolor and Pleurotus sajur-caju are well- was also investigated. Equilibrium studies were also
known white-rot fungi and strong degrader of various conducted with isotherm modeling. The heat-killed T.
xenobiotics. They could also be used to remove mercuric versicolor and P. sajur-caju were used to remove Hg(II)
ions from aqueous waters by adsorbing on its mycelium ions from synthetic wastewater samples.
(Kapoor et al., 1999).
In industrial or technical operations, immobilized
microbial cell systems could also provide additional
2. Experimental
advantages over freely suspended cells. These include
ease of regeneration and reuse of the biomass, easier
2.1. Micro-organism and media
solid–liquid separation and minimal clogging in con-
tinuous flow systems (Gadd and White, 1989; Zhou and
White-rot basidiomycete, T. versicolor and P. sajur-
Kiff, 1991). Natural polymers such as alginate, chitosan,
caju were maintained by subculturing on malt dextrose
chitin and cellulose derivatives have been mostly used as
agar slants. The growth medium was prepared using
the matrix for the immobilization of microbial cells via
deionized double-distilled water and subsequently filter
entrapment technique. These polymers are also known
sterilized; the final pH at 25 °C was adjusted to 4.5
to bind metal ions strongly (Ting and Sun, 1993; Arıca
(Table 1).
et al., 1993; Crist et al., 1994). Entrapment of microbial
Inocula were obtained from a seven day agar slant
cells in these polymer supports could also enhance mi-
culture. The fungal mat (0.3 g) was removed and mac-
crobial cell performance and adsorptive capacity of the
erated aseptically in 5.0 cm3 sterile medium using a ho-
biosorbent system for the heavy metal ions (Zhou and
mogenizer. It was used to inoculate 50 cm3 of medium in
Kiff, 1991; Ting and Sun, 1993; Arıca et al., 1993). Im-
250-ml flasks, and the flasks were incubated on a shaker
mobilized fungal cells were found to be far more stable
(150 rpm) for 5 days at 30 °C. After this period, the
during continuous operations in the bioreactor than the
fungal cells in free form.
Several researchers have reported that during heavy Table 1
metal biosorption using heat-killed or live cells, the Composition of growth medium
bioadsorptive capacity of the heat-killed cells might be Constituents Concentration (g dm
3 )
greater, equivalent to or less than that of living cell D -glucose 10.0
(Okino et al., 2000; Guibal et al., 1992). However, the KH2 PO4 20.0
MgSO4  7H2 O 0.5
use of heat-killed biomass in industrial application may
NH4 Cl 0.1
offer some advantages over living cells, such as less CaCl2  7H2 O 0.1
sensitivity to heavy metal ion concentration and adverse Thiamine 0.001
operating conditions (i.e., pH and temperature) (Okino Nitrilotriacetate 1.5
et al., 2000; Sa
g et al., 1995). NaCl 1.0
MnSO4  H2 O 0.5
Alginic acid is a heteropolysaccharide made of a-L -
FeSO4  7H2 O 0.1
glucuronic acid and b-D -manuronic acids and is found ZnSO4 0.1
in many algal species especially inside the brown algae. CaSO4 0.01
The overall composition and the sequence of monomers CuSO4  5H2 O 0.01
in the alginated polysaccharide vary extensively de- H3 BO3 0.01
NaMoO4  2H2 O 0.01
pending on the origin. This carboxylic polyelectrolyte is
 M.Y. Arıca et al. / Bioresource Technology 89 (2003) 145–154
biomass was harvested by filtration from the growth
medium and washed several times with distilled water.
The fungal biomass was obtained as discrete spherical
clumps; it was homogenized with a commercial blender
to destroy cell aggregates.
2.2. Immobilization of Trametes versicolor and Pleurotus
sajur-caju basidio spores
The immobilization of T. versicolor and P. sajur-caju
mycelium via entrapment was carried out as follows: Na-
alginate (2.0 g; from Macrosytia pyrifera, high viscosity,
Sigma Chem. Co., USA) was dissolved in distilled water
and then mixed with the fungal mycelium (50 cm3 , con-
taining 0.5 g fungal biomass). The mixture was intro-
duced into a solution containing 0.1 mol dm
3 CaCl2
with a burette and the solution was stirred to prevent
aggregation of the fungus entrapped Ca-alginate beads.
The fungus entrapped beads (4 mm) were cured in this
solution for 1 h and then washed twice with 200 cm3
sterile distilled water. The beads with immobilized myc-
elia were then transferred to the growth medium (50 cm3 )
in 250-cm3 flask and were incubated on an orbital shaker
(150 rpm) at 30 °C for 3 days. The mycelia growth in/on
the beads was followed during the incubation period by
using a microscope. After a 3-day incubation period, the
Ca-alginate beads with immobilized fungal mycelia were
removed from the medium by filtration and washed twice
with distilled water. This washed biomass is hereafter
called ‘‘immobilized live fungus’’. At times immobilized
live fungus was heated in 5 mmol dm
3 CaCl2 solution at
90 °C for 10 min; it will be referred to as ‘‘immobilized
heat-killed fungus’’. The immobilized preparations were
then stored at 4 °C in 5 mmol dm
3 CaCl2 solution until
use. The dry weight of the microbial growth in/on the
immobilized preparations was determined by weighing
(after drying in an oven at 50 °C overnight) the alginate
beads before and after cell growth.
2.3. Biosorption studies
The Ca-alginate and T. versicolor and P. sajur-caju
immobilized Ca-alginate beads were prepared by liquid
curing method in the presence of Ca(II) ions. Biosorp-
tion of Hg(II) ions onto plain alginate beads and onto
both immobilized live and heat-killed T. versicolor and
P. sajur-caju from aqueous solution was investigated in
batch biosorption-equilibrium experiments. The effects
of the medium pH and the initial concentration of
Hg(II) ions on the biosorption rate and capacity were
studied.
The effect of pH on the biosorption rate of the algi-
nate beads and both immobilized fungal preparations of
Hg(II) ions was investigated in the pH range of 3.0–7.0
(which was adjusted with HCl or NaOH at the begin-
ning of the experiment and not controlled afterwards) at
147
25 °C. Mercuric ion concentration in each solution (1.0
mmol dm
3 ) was prepared in distilled water (25 cm3 ) and
alginate, each live or heat-killed fungus entrapped in
alginate beads was transferred into biosorption medium
and agitated magnetically at 400 rpm.
The effect of temperature on the biosorption capacity
was studied between 15 and 45 °C at pH 5.5 with the
metal ion concentration of 1.00 mmol dm
3 .
The effect of initial Hg(II) ion concentration on the
biosorption was studied again at pH 5.5 and in the
concentration range of 0.150–3.00 mmol dm
3 .
2.4. Analytical procedure
Biosorption of Hg(II) ions from aqueous solutions
was studied in batch systems and the nitrate of mercury
ions was used. After the desired incubation period (about
120 min), the aqueous phases were separated from the
biosorbents and the concentration of Hg(II) ions in these
phases was determined. Throughout the study, a Shi-
madzu Model AA-6800 Atomic Absorption Spectro-
photometer (Japan) equipped with MVU-1 (Mercury
Vapor Unit) was used. Deuterium background correc-
tion was employed and the spectral slit width was chosen
as 0.5 nm. The optimized experimental conditions were
as below:
Working current 6.0 mA
Wavelength 253.6 nm
SnCl2 concentration 1% (w/v)
KMnO4 concentration 0.5% (w/v)
H2 SO4 concentration 5% (v/v)
For each set of data presented, standard statistical
methods were used to determine the mean values and
standard deviations. Confidence intervals of 95% were
calculated for each set of data in order to determine the
margin of error.
The amount of Hg(II) ions biosorbed per unit of Ca-
alginate and each fungus immobilized preparations (mg
metal ions/g dry beads) was obtained by using the fol-
lowing expression:
ðC0
CÞV
Q¼ ð1Þ
M
Where Q is the amount of Hg(II) ions adsorbed onto the
unit mass of the adsorbent (mg/g), C0 and C are the
concentrations of Hg(II) ions before and after biosorp-
tion (mg dm
3 ), V is the volume of the aqueous phase
(dm
3 ), and M is the mass of the adsorbent (g).
A known quantity of wet Ca-alginate or both fungus
immobilized preparations was used in the biosorption
experiments. After the biosorption process, the biosor-
bents were dried in an oven at 50 °C overnight and the
dry weight of the preparations was used in the calcula-
tions.
148 M.Y. Arıca et al. / Bioresource Technology 89 (2003) 145–154

2.5. Desorption/reuse ditions is also a very important parameter in the cell

In order to determine the reusability of the alginate
beads and both immobilized fungal preparations, con-
secutive adsorption–desorption cycles were repeated
three times by using the same biosorbents. Desorption
of Hg(II) ions was performed by using 10 mmol dm
3
HCl solution. The alginate and both immobilized fungal
preparations loaded with Hg(II) ions were placed in the
desorption medium and stirred at 400 rpm for 60 min at
25 °C. The final Hg(II) ion concentrations in the aque-
ous phase were determined by atomic absorption spect-
rometry. The desorption ratio was calculated from the
amount of metal ions adsorbed on the preparations and
the final Hg(II) ion concentration in the medium. The
following equation was used to calculate the desorption
ratio:
Amount of metal ions desorbed
Desorption ratio ¼
Amount of metal ions adsorbed
ð2Þ
Percent desorption values were obtained by multiplying
the above ratio by 100.
2.6. Wastewater studies
immobilization.
Representative SEM micrographs of the plain Ca-
alginate and fungus immobilized beads are presented
in Fig. 1a and b, respectively. The alginate beads are
spherical in shape with approximately 4 mm diameter.
The SEM micrograph of fungus immobilized alginate
bead was completely different from the empty one and it
showed an uniform fungal growth on the bead surface
indicating that the immobilized fungal mycelium is not
localized. This uniform distribution is an important
criterion for the proper biosorption of heavy metal ions
on the whole surface area of the fungus immobilized
beads. Thus, immobilization of fungal cells in/on the
alginate beads could also provide additional advantages
over the freely suspended fungal cells, in batch culture
fungal mycelia form individually distributed spherical
clumps (Ø 2–4 mm). This tight packing of the fungal
cells could also lead to diffusional restriction and less
adsorptive sites for heavy metals than the alginate-
fungal cells system.
The amount of entrapped fungus in the support was
about 0.15  0.01 g per gram of dry beads. It was de-
termined at the end of the three days cultivation period

Synthetic wastewater samples were prepared accord-

ing to European Union Directive 91/271/EEC, entitled
‘‘Concerning Waste Water Treatment’’. These sam-
ples contain 0.10 mmol dm
3 of Fe(III), Ni(II), Zn(II),
Cr(III) and Al(III) ions in 1000 mg dm
3 NaCl solution.
Hg(II) ion solution at 0.500 mmol/l concentration was
prepared by using synthetic wastewater and stirred with
heat-killed T. versicolor and P. sajur-caju at 400 rpm and
at pH 5.5 for 120 min and at a temperature of 20 °C.

2.7. SEM studies

Samples of Ca-alginate and fungus immobilized

beads were coated under vacuum with a thin layer of
gold and examined by scanning electron microscopy
(JEOL, Model JMS 5600).

3. Results and discussion

3.1. Properties of the alginate based biosorbent systems

Alginate is a natural polymer and can easily be con-

verted into hydrogels via crosslinking with divalent
calcium ions. It was preferred over other materials be-
cause of its various advantages such as biodegradability,
hydrophilicity, presence of carboxylic groups, and nat-
ural origin. The alginate beads were very stable over the
experimental pH range of 3.0–7.0. The operational sta- Fig. 1. The SEM micrographs of (a) plain Ca-alginate and (b) fungus
bility of the support under specified experimental con- immobilized beads.
 M.Y. Arıca et al. / Bioresource Technology 89 (2003) 145–154 149

and no fungal biomass increase was detected after this
period.
3.2. Biosorption of Hg(II) ions
3.2.1. Adsorption rate
Fig. 2 shows the changes in the amount of Hg(II) ions
biosorbed with time which was calculated by using Eq.
(1). Biosorption conditions are given in the figure leg-
end. The initial slope of the curve reflects the biosorp-
tion rate. It should be noted that no precipitation
occurred in these groups of experiments. High bio-
sorption rates were observed at the beginning, and then
plateau values (i.e., adsorption equilibrium) are gradu-
ally reached within 60 min.
3.2.2. Effect of initial metal ion concentration
Mercury ion biosorption capacities of the Ca-alginate
and both immobilized live and heat-killed two fungi
species are presented as a function of the equilibrium
concentration of Hg(II) ions within the aqueous bio-
sorption medium in Fig. 3. This figure was prepared by
using the plateau values of the biosorption rate-curves
(examples are given in Fig. 2).
The amount of Hg(II) ions adsorbed per unit mass
of the biosorbent (i.e., biosorption capacity) increased
with the initial concentration of metal ions, as expected.
It is well known that, at the beginning, the mass of metal
ion accumulated (per unit of cell mass) is directly pro-
portional to the concentration of the ion in the solution.
In order to reach the plateau values which represent
saturation of the active sites on the biosorbent, in other
terms to obtain the maximum biosorption capacity, the
initial concentration of Hg(II) ions was increased up to
3.00 mmol dm
3 . As seen in Fig. 3, the amount of Hg(II)
ions adsorbed on the plain alginate beads was 0.144 
0.005 mmol Hg(II)/g dry alginate beads. Maximum
biosorption capacity for immobilized live and heat-
killed fungal mycelia of T. versicolor was found to be
0.171  0.007 mmol Hg(II)/g and 0.383  0.012 mmol
Hg(II)/g, respectively; whereas for P. sajur-caju these
values were 0.450  0.014 mmol Hg(II)/g and 0.660 
0.019 mmol Hg(II)/g, respectively. The biosorption of
mercury on the immobilized heat-killed T. versicolor was
about 2.2 times higher than that of the immobilized live
form. This was 1.5 times higher for P. sajur-caju. The
increased mercury biosorption capacity may be due to
the changes in biosorptive characteristics of both fungal
species as a result of heat treatment. On the other hand,
both immobilized live and heat-killed P. sajur-caju
exhibited the highest Hg(II) biosorption capacity of
approximately 2.6 and 1.7 times, respectively, than that

Fig. 2. Adsorption rates of Hg(II) ions by (a) plain Ca-alginate and T. versicolor; (b) plain Ca-alginate and P. sajur-caju. Adsorption conditions:
initial concentration of Hg(II) ions: 200 mg dm
3 , pH: 5.5, temperature: 20 °C.
150 M.Y. Arıca et al. / Bioresource Technology 89 (2003) 145–154

Fig. 3. Biosorption capacities of (a) plain Ca-alginate and T. versicolor for Hg(II) ions; (b) plain Ca-alginate and P. sajur-caju for Hg(II)
ions. Biosorption conditions: amount of sorbent: 25 mg; volume of the biosorption medium: 25 cm3 ; pH: 5.5; temperature: 20 °C; biosorption time:
60 min.

of the T. versicolor counterparts. Difference between
fungal species in the magnitude of adsorption capacity
could be due to the difference in the cell wall structures
of these fungi (e.g. functional groups and surface area,
depending on fungal division, genera and species). These
functional groups such as amino, carboxylic, sulphydryl,
phosphate and thiol groups, differ in composition and in
their affinity for binding mercury.
Different sorbents (organic and biologic) having a
wide range of adsorption capacities for heavy metal ions
have been reported. Hafez et al. (1997) reported that the
biosorption capacity of Aspergillus flavus was 0.196
mmol for uranium per g of dry biomass. O € zer et al.
(1997) reported that the adsorption capacities of Rhi-
zopus arrhizus and the living E. coli strain were 0.354
mmol Hg(II)/g and 0.088 mg Hg(II)/g, respectively.
Various sorbent materials, other than microbial mass,
have also been used for removal of heavy metal ions from
industrial waste, ranging from natural polysaccharide
gels to coal and functionalised synthetic polymers. For
example, Shah and Devi (1996) used dithizone-anchored
poly(vinyl pyridine) support and they reported a specific
adsorption capacity up to 0.720 mmol/g. Liu et al. (1990)
achieved 0.359 mmol Hg(II)/g adsorption capacity with
N -hydroxymethyl thioamide resin. Cestari and Airold
(1997) found that the adsorption of mercury was 0.930
mmol Hg(II)/g by 3-trimethoxysilyl-1-propanethiol im-
mobilized silica. Say et al. (1998) used dithiocarbamate-
incorporated monosize polystyrene microspheres for
adsorption of organomercury species, where the maxi-
mum adsorption capacities were reported to be 0.609
mmol for CH3 HgCl, 0.570 mmol for C2 H5 HgCl and
0.100 mmol for C6 H5 HgCl per gram of the sorbent. All
these sorbents are expensive and require several steps
for preparation. The highest adsorption capacity of
0.660  0.019 mmol Hg(II)/g biosorbent obtained with
immobilized heat-killed P. sajur-caju was comparable
with the values reported in previous studies.
The modeling of the adsorption processes on Ca-
alginate and on both live and heat-killed fungi immo-
bilized systems was realized by applying different
adsorption isotherms. Among the several isotherm
equations, Langmuir and Freundlich adsorption iso-
therms were investigated, which are widely used to
analyze data for water and wastewater treatment ap-
plications.
The Langmuir equation is valid for monolayer
sorption onto a surface with a finite number of identical
sites and the model is described by the following equi-
librium:
 M.Y. Arıca et al. / Bioresource Technology 89 (2003) 145–154 151

k
M þ L ¼1 ML
k2
Metal ions and ligands on the biosorbents are denoted
by M and L, respectively, k1 and k2 are the forward and
reverse interaction rates which include the metal ion
movement from the bulk phase to the biosorbent surface
layer. Eq. (3) can be expressed in the form of a rate
equation with a second-order forward and a first-order
reverse kinetics, namely:
dq
¼ k1 Ceq ðqm
qeq Þ
k2 qeq
dt
Eq. (4) leads to
qm Ceq
qeq ¼
kd þ Ceq
where Ceq and qeq also show the residual metal concen-
tration and the amount of metal adsorbed on the ad-
sorbent at equilibrium, respectively, kd ¼ k2 =k1 is the
Langmuir constant of the system (Yang and Volesky,
1999; Esposito et al., 2001).
In addition, there may be interactions between ad-
sorbed molecules, a phenomenon referred to as cooper-
ativity. A molecule attached to a surface may make it
more or less difficult for another molecule to become
attached to a neighboring site and this would lead to a
deviation from the ideal adsorption equation. The em-
pirical Freundlich equation based on the amount of a
substance adsorbed ðqeq Þ is related to the concentration
Ceq by the equation:
qeq ¼ KF ðCeq Þ1=n
where KF and n are the Freundlich constants charac-
teristic of the system. KF and n are indicators of ad-
sorption capacity and adsorption intensity, respectively.
The slope and intercept of the linear Freundlich equa-
tion are equal to 1=n and KF , respectively.
Langmuir constants (qm and kd ) along with correla-
tion coefficients ðR2 Þ have been calculated from the plots
for biosorption of Hg(II) ions on the biosorbents and
the results are presented in Table 2. The maximum
capacity qm determined from the Langmuir isotherms
defines the total capacity of the biosorbents for Hg(II)
ions.
ð3Þ The magnitudes of KF and n (Freundlich constants)
show easy separation of metal ions from aqueous me-
dium and indicate favourable adsorption. The intercept
KF is an indication of the adsorption capacity of the
adsorbent; the slope 1=n shows the effect of concentra-
tion on the adsorption capacity and represents adsorp-
tion intensity. The magnitude of KF and n values (Table
2) showed easy uptake of Hg(II) ions from the aqueous
medium with a high adsorption capacity for immobi-
lized live and heat-killed fungus. For all experimentally
tested biosorbents, n values were found high enough for
ð4Þ separation.
3.2.3. Effect of pH and the temperature on the biosorption
capacity of the biosorbents
ð5Þ
It is well known that metal ion adsorption on both
non-specific and specific sorbents is pH dependent
(Fourest and Roux, 1992; O € zer et al., 1997). The me-
dium pH affects the solubility of metal ions and the
ionisation of the functional groups (i.e., carboxylate,
phosphate, and amino groups) on the fungal cell wall
(Gadd and White, 1989; Shah and Devi, 1996). Fig. 4
shows the effect of pH on biosorption. Biosorption of
the Hg(II) ions first increased with pH, and maximum
Hg(II) ions biosorption occurred between pH 4.0 and
6.0. For the interaction of the Hg(II) ions with the al-
ginate and immobilized fungal cell wall the responsible
component could be primarily the carboxylate groups
on both alginate and the cell wall component of the
mycelia. It has been proposed that the groups respon-
sible for metal binding are carboxyl groups, which have
ð6Þ pKa Õs between 3 and 4 (Okino et al., 2000). The increase
in binding that occurred at pH 4.0 can be attributed to
these deprotonated and negatively charged species.
The temperature of the adsorption medium could be
important for energy dependent mechanisms in metal
biosorption by microorganisms. Energy-independent
mechanisms are less likely to be affected by temperature
since the process responsible for biosorption is largely
physicochemical in nature. The biosorption of Hg(II)
ions by alginate and both immobilized live and heat-
killed fungus appeared to be independent of temperature
over the temperature range tested (15–45 °C). Similar
results were reported by other researchers (Okino et al.,
2000; Gadd and White, 1989).

Table 2
Isotherm model constants and correlation coefficients for biosorption of Hg(II) ions from aqueous solution
Biosorbent Langmuir Freundlich
qm (mg/g) Kd M ð 107 Þ R2 KF n R2
Ca-alginate 31.70 3.69 0.995 4.12 2.89 0.981
Live T. versicolor 43.49 6.99 0.999 0.98 1.62 0.999
Heat-killed T. versicolor 103.9 8.46 0.998 1.68 1.52 0.995
Live P. sajur-caju 138.89 0.16 0.999 0.97 1.33 0.996
Heat-killed P. sajur-caju 172.41 7.84 0.999 4.29 1.72 0.993
152 M.Y. Arıca et al. / Bioresource Technology 89 (2003) 145–154

Fig. 4. Effect of pH on the biosorption capacities of (a) Ca-alginate and T. versicolor fungus for Hg(II) ions; (b) Ca-alginate and P. sajur-caju fungus
for Hg(II) ions. Biosorption conditions: initial concentration of Hg(II) ions: 200 mg dm
3 ; amount of polymer: 25 mg; volume of biosorption
medium: 25 cm3 ; temperature: 20 °C; biosorption time: 60 min.

3.2.4. Desorption and reuse
The desorption of the adsorbed Hg(II) ions from
the biosorbents was studied in a batch system. Hg(II)
ions adsorbed onto biosorbents were eluted with 10
mmol dm
3 HCl. More than 97% of the adsorbed Hg(II)
ions was desorbed from the biosorbents. In order to
show the reusability of the biosorbents; adsorption–
desorption cycle of Hg(II) ions was repeated five times
by using the most effective form of the biosorbents, i.e.,
immobilized heat-killed T. versicolor and P. sajur-caju.
The adsorption capacities of the biosorbents did not
noticeably change (only a maximum 3% change was
observed with the tested biosorbent) during the repeated
adsorption–desorption operations (Fig. 5). These results
showed that inactivated fungus entrapped biosorbents
could be repeatedly used in Hg(II) ion adsorption
studies without significant losses in their initial adsorp-
tion capacities.
3.2.5. Removal of Hg(II) ions from synthetic wastewater
In this group of experiments, removal of Hg(II) ions
from synthetic wastewater by heat-killed T. versicolor
and P. sajur-caju was studied. Synthetic waste water
samples were prepared according to European Union
Directive 91/271/EEC, entitled ‘‘Concerning Waste
Water Treatment’’. These samples contained 0.10
mmol dm
3 of Fe(III), Ni(II), Zn(II), Cr(III) and Al(III)
ions in 1000 mg dm
3 NaCl solution. Hg(II) ion solution
at 0.500 mmol dm
3 concentration was prepared by
using synthetic wastewater and interacted with heat-
killed T. versicolor and P. sajur-caju for 120 min at pH
5.5 and at a temperature of 20 °C. Fig. 6 depicts that
in about 60 min 73% and 81% of Hg(II) ions present
initially were removed by heat-killed T. versicolor and
P. sajur-caju, respectively.
4. Conclusion
T. versicolor and P. sajur-caju were entrapped into
alginate beads in three days growth period. The live and
heat inactivated forms have been successfully used as the
biosorbing agent for removal of Hg(II) ions from
aqueous medium by using plain of the alginate beads as
a control system. The biosorption of Hg(II) ions on all
the tested fungal preparations depends on the experi-
mental conditions, particularly, medium pH and the
concentration of mercury ions in the medium. The
biosorption medium temperature had no significant ef-
fect on the biosorption capacities of all the biosorbents
 M.Y. Arıca et al. / Bioresource Technology 89 (2003) 145–154 153

Fig. 5. Adsorption/desorption cycles for (a) heat-killed T. versicolor; (b) heat-killed P. sajur-caju.

Fig. 6. Removal of Hg(II) ions from synthetic wastewater by heat-killed T. versicolor and P. sajur-caju.

between 15 and 45 °C. The adsorption equilibrium was
well described by Langmuir and Freundlich adsorption
isotherms for all the tested biosorbents. The heat in-
activated preparations of T. versicolor and P. sajur-caju
showed improved performance in the batch system over
those of the active immobilized forms, especially, the
heat inactivated preparation of P. sajur-caju could be
used as an efficient biosorbent system for the treatment
of heavy metal containing wastewater streams. These
biosorbents can be regenerated and reused by acid
treatment. The immobilized heat-inactivated T. versi-
color and P. sajur-caju were reused in five biosorption
and desorption cycles with negligible decrease (upto 97%
recovery) in their biosorption capacities. The non-living
immobilized fungal preparation also eliminates the
problem of metals toxicity, the possible adverse oper-
ating conditions in packed bed and continuous flow
systems.
154 M.Y. Arıca et al. / Bioresource Technology 89 (2003) 145–154
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