Metal biosorption by PAN-immobilized fungal biomass insimulated wastewaters
Anastasios I. Zouboulis a,�, Kostas A. Matis a, Maria Loukidou a,Ferdinand Sebesta b
a Chemical Technology Division, School of Chemistry, Aristotle University of Thessaloniki, GR-540 06 Thessaloniki, Greeceb Department of Nuclear Chemistry, Czech Technical University, Prague, Czech Republic
Received 4 January 2002; accepted 13 June 2002
Abstract
The ability of microorganisms to remove metal ions from solution was investigated by using dead fungal biomass.
The latter was immobilized by polyacrylonitrile, a known binding polymer for inorganic ion exchangers. This product
was examined in batchwise experiments for the removal of toxic metals from aqueous mixtures containing copper, zinc
and nickel (i.e. simulated wastewaters) in order to examine whether this separation technique may improve biomass
performance as a metals sorbent. The metal removal capacities of the beads PAN-B/50% for zinc, copper and nickel
were 16, 7 and 0.25 mg g�1, respectively, while the zinc, copper and nickel adsorption capacities of PAN-B/75% were
18, 7.9 and 0.25 mg g�1, respectively. The obtained results were compared with those using plain dispersed biomass.
Certain column experiments were also performed. Promising results were obtained in the laboratory, as effective metal
removals were observed.
# 2002 Elsevier Science B.V. All rights reserved.
Keywords: Biosorption; Immobilization; Polyacrylonitrile; Fungi; Metals
1. Introduction
Strict environmental protection legislation and
public environmental concerns leads chemical
industry to invest intensively for innovate technol-
ogy that protects environment and cleans up
existing waste sites, as it was reported in a recent
report dealing with the current trends in chemical
technology [1]. Ongoing concern about the pro-
duction of chemicals and their fate in the environ-
ment will drive further developments towards
fewer, less dangerous by-products or by-products
that can be recycled or disposed without creating
secondary problems. Environmental chemistry
may become, according to the American Chemical
Society, the most visible technology with a multi-
tude of growth areas. Any application that helps
to reduce pollutants to the environment will
command in the near future a prominent place.Dissolved heavy metals escaping into the envir-
onment may pose finally a serious health hazard.
� Corresponding author. Tel.: �/30-3199-7794; fax: �/30-
3199-7759
E-mail address: [email protected] (A.I. Zouboulis).
Colloids and Surfaces A: Physicochem. Eng. Aspects 212 (2003) 185�/195
www.elsevier.com/locate/colsurfa
0927-7757/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved.
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Controlling heavy metal discharges and removing
toxic metals from wastewaters have become a
serious concern, especially for the chemical indus-
try. Conventional methods for heavy metal re-
moval are mainly precipitation and less extensively
used evaporation or ion exchange. As these
methods are becoming more inadequate and
costly, environmental technology investigates
other alternatives, in terms of efficiency, practi-
cality and particularly, economical applications.
Biosorption of toxic metals using microorganisms
has received a great deal of attention in recent
years not only as a novel treatment process, but
also for its potential application in industry. Metal
accumulative bioprocesses generally are divided
into two categories, biosorptive (passive) uptake
by non-living biomass and bioaccumulation by
living cells [2]. Maintaining a viable biomass
during the metal removal process can be very
difficult. Therefore, biosorption utilizing the abil-
ity of non-living biomass to accumulate heavy
metals from wastewater is considered as a more
competitive, effective and economical attractive
method [3].
Many of the recent studies on metals biosorp-
tion have been focused to single solutions, contain-
ing one metal and only in limited cases; the
influence of other cations on the overall metal
uptake process has been reported. It is known that
the co-existence of metal ions in most wastewaters
can affect their sorption of metal ions to the
biomass and may interfere to some extent with
the observed removal efficiency. Metal ions may
interact to a synergistic, antagonistic or non-
interactive manner [4]. This fact has emphasized
the need for studying the behavior of metal ions
mixtures during biosorption.
The environmental pollution of toxic metals is
spreading throughout the world along with indus-
trial progress; zinc, copper and nickel are known
to be commonly used heavy metals and they are
some of the more widespread metal contaminants
of the environment [5]. Traces of these kinds of
metals are also necessary as cofactors of enzymatic
reactions, but high levels of them could be
extremely toxic to living organisms, whereas
metabolic reactions can be inhibited.
Previous studies carried out using the biomassof Penicillium chrysogenum , as freely suspended
biomass, for the removal of Zn, Cu and Ni from
an aqueous mixture, indicated that there was a
preferential uptake of copper, which was removed
at a higher level than nickel or zinc [6]. Nickel and
zinc removals were most affected by the presence
of calcium and sodium ions, due to the fact that
for these metals carboxyl groups are the mainligands and hence, the competitive effect was
found to be greater. When using dispersed sys-
tems, there is a need for a subsequent solid�/liquid
separation stage, prior to regeneration of biomass
and recovery of metal. Dispresed-air flotation
(using dodecylamine as collector) was successfully
applied for the effective biomass separation. Dur-
ing repetitive cycles of biosorption�/flotation�/elu-tion process, low elution of metals was observed,
while there was a need for additional surfactant, a
fact that possibly increases the supplementary
process cost [6].
It has been recognized that immobilizing bio-
mass in a polymer matrix may improve the biomass
performance and capacity and simultaneously
there is not need for the solid�/liquid separationof metal-laden biomass from the treated solution.
Immobilization allows also column operation.
Various techniques were previously published
and applied to metals removal, such as: Chlorella
homosphaera immobilized by sodium alginate [7];
Sargassum fluitans and Ascophyllum nodosum
crosslinked with formaldehyde, glutaraldehyde or
embedded in polyethylene imine [8]; sphagnumpeat moss immobilized in porous polysulphonate
beads [9]; and Rhizopus arrhizus immobilized in
reticulated polyurethane foam [10]. In the present
paper, modified polyacrylonitrile (denoted here-
after as PAN) was used as binder of fungal biomass
(dead cells of P. chrysogenum , a waste by-product
during antibiotic production). This biomass was
obtained after thermal treatment (sterilization).PAN was previously used as a convenient
binding polymer for a number of inorganic ion
exchangers; the resulting sorbents have been tested
for the separation and preconcentration of differ-
ent contaminants, including radioactive wastes
[11]. The kinetics of ion exchange and the sorption
capacity of these composite materials were not
A.I. Zouboulis et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 212 (2003) 185�/195186
influenced by the binding polymer, whereas theirphysico-chemical properties (as hydrophilicity,
mechanical strength etc.) can be modified by the
degree of cross-linking of the polymer, the use of
suitable co-polymers or by changing the composi-
tion and temperature of polymerization. These
materials can be efficiently used in column packed
beds. The techniques of preparation inorganic ion-
exchanger granules, as well as methods for im-provement the granular strength of these materials
were recently reviewed [12].
After mixing the binding polymer with the
biomass in this investigation, the entrapped fungal
cells were used for the removal by biosorption of
Zn(II), Cu(II) and Ni(II) cations from aqueous
mixtures in a batch system. The biosorption
characteristics and performance of PAN�/Mycanbeads as a function of flow rate and feed concen-
tration was also tested in a packed-bed column
configuration.
2. Materials and methods
2.1. Biomass
The used fungal non-living biomass was P.
chrysogenum , an industrial waste with trade
name Mycan, supplied by Synpac Ltd. (UK)
through the University of Newcastle-upon-Tyne,
Department of Microbiology. It had a final water
content of around 7%. The sample was extensively
washed by deionized water for the removal ofvarious soluble residuals and dried in room
temperature (25 8C). Aqueous suspensions
showed a natural pH around 6. The pore volume
and the specific surface area of biomass were
determined with a Quantachrome Autosorb appa-
ratus, applying nitrogen adsorption and deso-
rption.
2.2. Biosorbent preparation
The preparation of adsorbent beads using poly-
acrylonitrile, which act as organic binding matrix,
was performed in the Department of Nuclear
Chemistry, Technical University of Prague (Czech
Republic). The Mycan was dispersed in a solution
of matrix�/matrix�/monomer�/matrix�/componentin a water bath, followed by coagulation, poly-
merization and polycondensation of the dispersion
and then washed with water; the separation of
obtained product was accomplished by wet sieving
[13]. The content of biomass in the product can be
varied over a broad range. The procedure can be
simplified or modified in several ways. The final
granules can be also produced in different forms,as desired [12]. Two different samples, in pseudo-
spherical form were prepared and examined ex-
perimentally containing varying biomass (the ac-
tive component) concentrations, P??-?50% and
P??-?75%, respectively. The grain size was between
0.8 and 1.44 mm.
2.3. Biosorption
Biosorption studies were performed in two
subsequent cycles (following elution), in conical
flasks stirred for 24 h in a reciprocal shaker (at
180�/190 rpm) at ambient temperature (20�/
25 8C). The elution was conducted at a solid/
liquid ratio of 25:1 (mg of biomass per ml of
solution) and it was performed using a membrane
vacuum filter as published elsewhere [14]. Theapplied eluant for the desorption of metals from
the loaded sorbent was a mixture of sodium
sulphate (1 M) and tri-sodium citrate (0.25 M).
An aqueous mixture of metals, commonly found
in many industrial wastewaters was prepared, from
the respective nitrate salts, containing Zn, 50; Cu,
10; Ni, 2 (mg l�1), as well as Ca, Mg, Na each
equal to 100 mg l�1. The presence of last threemetals, although not presenting any interest for
their removal, was examined in order to investigate
the respecting interference caused by their pre-
sence. Blank experiments (without the presence of
biomass) were also carried out. The obtained
results with the immobilized biomass were also
compared with those found, using plain dispersed
biomass. The solution pH was adjusted initially bythe addition of HNO3 or NaOH solutions. The
metal solution was 150 ml and the concentration of
immobilized fungal biomass was 1 g l�1.
Column tests were carried out using PAN-B75%
biomass (5 g l�1), at a pH value of 7 and treating
the same mixture of metals. The mixture was
A.I. Zouboulis et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 212 (2003) 185�/195 187
passing through the bed of adsorbent beads at apumping rate of 470 ml h�1, with the assistance of
a peristaltic pump (porosity, 0.5 g, linear velocity
0.7 m h�1).
The residual concentrations of zinc, copper and
nickel were analyzed by Atomic Absorption Spec-
trophotometry, after separation of used sorbent by
filtration. The percentage removal of metals re-
lative to the initial amount (RE%) was recorded.
3. Results and discussion
A barrier often mentioned in current relative
research is the difficulty in tailoring adsorbent
materials to handle complex streams [15]. It was
advocated that process improvements would be
needed to take full advantages of new adsorbentmaterials. Therefore, the experience obtained pre-
viously with composite ion exchangers [11] was
applied in this work for the biosorption process.
For the calculation of monolayer capacity, the
Brunauer�/Emmett�/Teller equation was applied,
following the standard adsorption procedure [16].
The specific surface area of beads was calculated
from the nitrogen adsorption curve as 9.7 m2 g�1
(Fig. 1(a)), which is much smaller than most
typical adsorbents. The pore volume calculated
from the desorption curve was found to be 0.016
cm3 g�1 (Fig. 1(b)). Taking into account that the
specific surface area and the pore volume were
rather small, the pore size distribution was not of
any significant importance.
The possibility of applying PAN-biomass gran-ules as metal adsorbents can be illustrated and
compared with the reference samples in Fig. 2. The
removal of zinc, nickel and copper ions were quite
comparable with that obtained by using freely
suspended biomass. Although the removals were
found somewhat lower, consideration should be
taken here of the relatively smaller overall biomass
concentration, contained in the immobilized sam-ples. Specific preference for copper removal over
zinc and nickel from the multicomponent solution
can be observed. Similar observations were re-
ported in other related studies on metal sorption
from multimetal solutions, using a biomass of
Streptomyces noursei , where the removal of ca-
tions showed an order of Cu2��/Zn2��/Ni2�
[17]. Elsewhere [18] it was reported also that the
amount of Cu2� bound by Chlorella regularis was
higher than the amounts of Zn2� or Ni2�. It has
been found by Cabral [19] using Pseudomonas
syringae that the metals can be classified into three
categories on the basis of the extent of metal
biosorption from a multicomponent solution: high
affinity (e.g. Pb), intermediate affinity (e.g. Cd, Cuand Zn) and low affinity (e.g. Ni and Co).
The presence of another solute (metal cation)
can interfere the biosorption of desired metal by
competing for similar binding sites on the cell
surface, such as amine, carboxylate, phosphate,
imidazole etc. functional groups. Inhibitory effects
of divalent cations on biosorption have been
reported for R. arrhizus [20]. Also, Ahuja et al.[21] have found that the inhibitory effect of Mg2�
was much stronger than that of Ca2� during zinc
biosorption. However, cadmium uptake by Toly-
pothrix tenius was not strongly affected by the
presence of Mg and Ca ions [22]. Heavy metals
removal by ion-exchange resins is generally sensi-
tive to the presence of coexisting Ca2�, Mg2� and
Na� cations, whereas the fungal biomass does notseem to adsorb them in a great extent [23]. Thus,
the use of entrapped fungal biomass in PAN beads
may be advantageous over the convenional ion-
exchange resins, when Ca2�, Mg2� and Na�
cations are also present in industrial wastewaters
at relatively high concentrations.
Metal ion adsorption on both non-specific and
specific sorbents is pH dependent [24]. The pHaffects the availability of metal ions in solution
(speciation) and the metal binding sites as cell
surface. In a previous relative electrokinetic study
[6], it was found that the zeta-potential of fungal
cell wall (being initially anionic in nature) was
influenced by pH and hence, the sorption behavior
of metals was changed.
Acidic conditions did not favor the sorption ofcations. The poor sorption of metals in the low pH
range could be devoted to competition with H�
ions for the same metal binding sites on the
bacterial cell. Nevertheless, by increasing the
solution pH, there is an increase of ligands
carrying negative charges, which results in in-
creased binding of cations. Significant improve-
A.I. Zouboulis et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 212 (2003) 185�/195188
ment in each metal sorption was observed over the
pH value of 7 and this trend continued and
reached a maximum removal at approximately
pH 10. Nevertheless, it has to be mentioned that at
alkaline region, metals precipitate as insoluble
hydroxides onto the cell surfaces. It is known
(using the MINEQL�/ program) that Cu precipitates
as hydroxide at pH of about 6, followed by Zn at
pH 7.3, while Ni precipitates at pH of 8 (Fig. 3).
Further, from the application point of view,
precipitation may desirably augment and act
complimentary with the application of biosorption
to achieve the required high removal of metals.
Selected elution results of metals from metal-
laden biomass are presented in Fig. 4. The
recovery of metals previously sorbed onto biomass
is one of important aspects for the successful
development of biosorption [25]. In the present
study, a mixture of sodium sulphate and tri-
sodium citrate proved quite effective to desorb
Fig. 1. (a) BET plot for the sorption onto the plain biomass; 1/[W((Po/P)�/1)]: is the amount of nitrogen adsorbed and (b) cumulative
pore volume from desorption data.
A.I. Zouboulis et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 212 (2003) 185�/195 189
Fig. 2. Effect of pH on the removal of Zn, Cu and Ni applying two PAN�/biomass beads with different biomass content; Cres is the
residual concentration of metals (note the different initial concentration of them).
A.I. Zouboulis et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 212 (2003) 185�/195190
Cu and Zn, with an average efficiency 80 and 60%,
respectively, while for nickel the percentage deso-
rption was restricted to only 15%. Mineral acids,
being quite effective in metal desorption, may
inflict cell damage [26] and render the biomass
unsuitable for reuse [27]. Calcium carbonate (10
mM) was found more efficient for Ni desorption
(71%), as compared with Cu (57%), as this reagent
is more specific for Cu removal (88%) than Ni
(21%) from P. aeruginosa [28].When testing the two bead samples in a second
biosorption cycle, following elution, promising
results were obtained, as presented in Fig. 5. The
adsorptive capacity was not highly affected. It was
also observed that shorter retention times during
desorption were resulting in lower elution of
metals and therefore, in lower metal removals
during the second cycle. It is worth noting,
however, that this way of expression for metal
removals may be misleading, due to the different
initial concentrations of them in the aqueous feed
(zinc was present at five times higher concentra-
tion, as compared with copper).
The comparison between the two operation
cycles (Fig. 5) showed that the removal of metals
was increased with pH. Maximum percentage
removals for Cu(II) and Zn(II) cations occurred
between pH 9�/10, where nickel was almost
completely removed (�/100%). The above satis-
factory metal removals were recorded for the
PAN-fungi biomass (75%). The higher nickel
removal during the second cycle was possibly due
to different biosorption mechanism. It is known
that mechanism may involve microprecipitation of
the metal or the metal penetration through the cell
wall [29]. The nickel ion compared with other
heavy metal ions, was a more recalcitrant pollu-
tant and different strains of microorganisms have
a relatively low Ni-binding capacity [30]. This was
probably due to specific chemical properties of
nickel ions leading to steric hindrance of biosorp-
tion. Nickel, according to Pearson classification,
belongs to the borderline class presenting inter-
mediate coordination behavior. So, nickel biosorp-
tion, which is generally low, is strongly depressed
in the presence of other elements.
However, certain problems were encountered
when the other physical characteristics of PAN
dispersion were examined, showing increased tur-
bidities, after a longer (48 h) contact time (Figs. 6
and 7). An average loss of 10% weight was noticed
also for the PAN-based samples, in both cycles.
Similar observation of weight loss (28% for six
cycles) of immobilized biomass, due to mechanical
attrition, was reported also for a sheet bioreactor
[10]. In order to eliminate the biosorbent losses,
further modification changes have to be applied
during the preparation technique, in order to
obtain higher resistant granules to the attrition
forces of the system. The chemical and radiation
stability of the composite sorbents using PAN
were elsewhere examined and the stability of PAN
Fig. 3. Speciation diagrams of (a) Zn, (b) Cu and (c) Ni
predicted by MINEQL.
A.I. Zouboulis et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 212 (2003) 185�/195 191
binder was found to be limited in alkaline solu-
tions [12].
In columns treating multicomponent mixtures,
heavy metal ions compete for a certain number of
binding sites in the biomass. Depending on the
composition of wastewater and the form of
biomass, competitive ion exchange may severely
reduce the efficiency of this metal removal process
[31]. In the present study, the metal solution was
passed through an adsorption column. A lag time
was observed for metals to reach and interact with
the binding sites on the cell surface. Therefore, by
increasing the residence time the possibility of
contact between metals and binding sites would be
enhanced, but long residence time may lead to
undesirable desorption. The best performance was
achieved in approximately 1 h using these experi-
mental conditions. Nearly 100% removal of Cu
ions was obtained at pH 7, as shown in Fig. 6,
while the removal efficiency of nickel and zinc was
decreased dramatically to 20% with time, as the
binding sites became faster saturated (after passing
1.5 l of metal mixture). The experimental data
indicated that biosorption process is quite effective
in removing high concentration of copper ions
from multicomponent aqueous solution.
4. Conclusions
The simultaneous presence of different metals
ions during biosorption environment can have
significant consequences in the process efficiency.
Since a real wastewater contains often a lot of
pollutants, multicomponent systems need to be
further studied. In this initial study, the ability of
P. chrysogenum to bind (and remove) three metals,
i.e. Cu2�, Zn2� and Ni2�, in the presence of co-
existing Na�, Ca2� and Mg2� from aqueous
mixtures was investigated. To overcome the se-
paration problems of using dead biomass in
powdered form, as well as mass loss after regen-
eration and small particle size, the biomass was
immobilized in a polymer matrix.Biosorption studies of PAN�/P. chrysogenum
beads have been found to be effective in removing
relatively low concentrations of three commonly
found heavy metals from wastewaters. The process
was mainly influenced by pH and immobilization
procedure. At pH 7, the removal capacities of the
P??-?50% beads were for 16 mg Zn g�1, 7 mg Cu
g�1 and 0.25 mg Ni g�1, while the removal
capacities obtained for PAN-B/75% beads were
for zinc 18, copper 7.9 and nickel 0.25 mg g�1.
Fig. 4. Elution of metals following the 1st biosorption cycle from PAN-B75%.
A.I. Zouboulis et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 212 (2003) 185�/195192
The adsorbed metals can be quite effectively
eluted; the beads can be regenerated and then
reused. Biosorption of heavy metals by immobi-
lized fungal biomass in a packed bed column is an
economical and feasible technology for removing
metal ions. Packed bed adsorption has a number
Fig. 5. Comparison of the first and the second cycle, for the removal of metals: (a) PAN-B50%, and (b) PAN-B75%; Cres is the residual
concentration of metals.
A.I. Zouboulis et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 212 (2003) 185�/195 193
of advantages, such as its simplicity to operate and
it may be easily scaled up from a laboratory-scale
procedure.
The present work showed that immobilized
biomass of P. chrysogenum appears as a new
possible biosorbent granule to be used for treat-
ment of metal-bearing solutions.
Acknowledgements
Thanks are due to E.G. Rousou, Chemist, for
experimental collaboration, to CPERI, Technolo-
gical Park of Thessaloniki, where the porosity
measurements of the material were conducted and
to Dr I.C. Hancock at the Microbiology Depart-
Fig. 6. Properties of the immobilized bead PAN-B50%: weight loss and turbidity with pH.
Fig. 7. Column biosorption test with PAN-B75%. Condition pH: 7, linear velocity: 0.8 m h�1; bed volume is the ratio between the
treated volume and volume of the column bed (adimentional).
A.I. Zouboulis et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 212 (2003) 185�/195194
ment, University of Newcastle-upon-Tyne, UK,where the biomass was prepared.
References
[1] ACS, Current Trends in Chemical Technology, Business
and Employment, American Chemical Society, Washing-
ton, 1998, p. 20.
[2] B. Volesky, H. May, Z.R. Holan, Biotechnol. Bioeng. 41
(1992) 826.
[3] A.I. Zouboulis, K.A. Matis, I.C. Hancock, Sep. Purif.
Methods 26 (1997) 255.
[4] Y.P. Ting, W.K. Teo, Biores. Technol. 50 (1994) 113.
[5] G. Donmez, Z. Aksu, Process Biochem. 35 (1999) 135.
[6] A.I. Zouboulis, E.G. Rousou, K.A. Matis, I.C. Hancock,
J. Chem. Technol. Biotechnol. 7 (1999) 1.
[7] A.C.A. Costa, S.C.F. Leite, Biotech. Lett. 13 (1991) 559.
[8] A. Leusch, Z.R. Holan, B. Volesky, J. Chem. Technol.
Biotechnol. 62 (1995) 279.
[9] E.M. Trujillo, T.H. Jeffers, C. Ferguson, H.Q. Stevenson,
Environ. Sci. Technol. 25 (1991) 1559.
[10] R. Ileri, A. Akkoyunlu, Fresenius Environ. Bull. 8 (1999)
397.
[11] F. .Sebesta, J. John, A. Motl, Waste Treatment and
Immobilization Technologies Involving Inorganic Sor-
bents, Tecdoc 947, IAEA, 1997, p. 79.
[12] F. .Sebesta, J. Radioanal. Nucl. Chem. 220 (1997) 77.
[13] F. Sebesta, Chech Patent (1992) AO 273369.
[14] T.J. Butter, L.M. Evison, I.C. Hancock, F.S. Holland,
K.A. Matis, A. Philipson, A.I. Sheikh, A.I. Zouboulis,
Water Res. 32 (1998) 400.
[15] S. Adler, E. Beaver, E. Bryan, J.E.L. Rogers, S. Robinson,
C. Russomanno, Vision 2020: Separations Roadmap,
Center for Waste Reduction Technologies, AIChE, New
York, 1998, p. 15.
[16] K.W. Sing, in: A.E. Rodrigues, M.D. LeVan, D. Tondeur
(Eds.), Adsorption: Science and Technology, Kluwer
Academic Press, Dordrecht, 1989, p. 3.
[17] B. Mattuschka, K. Junghans, G. Straube, in: A.E. Torma,
M.L. Apel, C.L. Brierley (Eds.), Biohydrometallurgical
Technology, vol. II, Mineral Metals and Materials Society,
Warrendale, PA, 1993, p. 125.
[18] A. Nakajima, T. Hosikoshi, T. Sakagushi, Eur. J. Appl.
Microbiol. Biotechnol. 12 (1981) 76.
[19] J.P.S. Cabral, Microbios 71 (1992) 47.
[20] D. Lewis, R.J. Kiff, Environ. Technol. Lett. 9 (1988) 991.
[21] P. Ahuja, R. Gupta, R.K. Saxena, Curr. Microbiol. 35
(1997) 151.
[22] D. Inthorn, H. Nagase, Y. Isaji, K. Hirata, K. Miyamoto,
J. Ferment. Bioeng. 82 (1996) 580.
[23] J.M. Tobin, D.G. Cooper, R.J. Neufeld, Appl. Env.
Microbiol. 47 (1994) 821.
[24] A. Kapoor, T. Viraraghavan, D.R. Cullimore, Biores.
Technol. 70 (1999) 95.
[25] B. Volesky, Trends Biotechnol. 5 (1987) 96.
[26] M. Tsezos, Biotechnol. Bioeng. 26 (1984) 973.
[27] C.Z.M. Hu, M.J. Norman, D. Faison, E. Reeves, Biotech-
nol. Prog. 13 (1996) 60.
[28] P. Sar, S.K. Kazy, R.K. Asthana, S.P. Singh, Intern.
Biodet. Biodegr. 44 (1999) 101.
[29] M.N. Hughes, R.K. Poole, Metals and Microorganisms,
Chapman & Hall, London, 1989, p. 328.
[30] M. Tsezos, E. Remoudaki, V. Angelatou, Intern. Biodet.
Biodegr. 38 (1996) 19.
[31] D. Kratochvil, B. Volesky, Water Res. 34 (2000) 3186.
A.I. Zouboulis et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 212 (2003) 185�/195 195