metal biosorption by pan-immobilized fungal biomass in simulated wastewaters

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.

PII: S 0 9 2 7 - 7 7 5 7 ( 0 2 ) 0 0 3 0 4 - 7

mailto:[email protected]

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-


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.


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).


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.


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%.


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.


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).


ment, University of Newcastle-upon-Tyne, UK,where the biomass was prepared.

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metal biosorption by pan-immobilized fungal biomass in simulated wastewaters

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