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Water Research 39 (2005) 2273–2280

www.elsevier.com/locate/watres

Metal ion adsorption by Phomopsis sp. biomaterial inlaboratory experiments and real wastewater treatments

Filippo Saianoa,�, Maurizio Ciofaloa, Santa Olga Cacciolab, Stefania Ramireza

aDipartimento ITAF, Sezione Chimica, Universita di Palermo, Viale delle Scienze, Edificio 4, I-90128 Palermo, ItalybDipartimento S.EN.FI.MI.ZO., Sezione di Patologia Vegetale e Microbiologia Agraria, Universita di Palermo, Viale delle Scienze,

Edificio 5, I - 90128 Palermo, Italy

Received 14 April 2004; received in revised form 10 March 2005

Available online 6 June 2005

Abstract

An insoluble material of polysaccharidic nature has been obtained by thermal alkali treatment of the filamentous

fungus Phomopsis sp. FT-IR spectrum of the resulting material as well as its nitrogen content suggest that chitosan and

glucans are the main components of the biomaterial. Information on Lewis base sites has also been obtained and used

as a guideline in the evaluation of the complexing ability against a number of metal ions in aqueous media at pH in the

range 4–6. Results indicate that after 24 h contact time, up to 870 mmol/g of lead, 390mmol/g of copper, 230 mmol/g ofcadmium, 150 mmol/g of zinc and 110 mmol/g of nickel ions are adsorbed into the material. After approximately 10min,about 70% of the overall adsorption process has already been completed. Adsorbed metal ions can be recovered by

washing with dilute acid. Experiments have been extended to a real wastewater effluent confirming the potential of this

biomaterial as a depolluting agent.

r 2005 Elsevier Ltd. All rights reserved.

Keywords: Fungal biomaterial; Chitin; Chitosan; Adsorption isotherm; Heavy metal; Wastewater

1. Introduction

A number of physico-chemical methods such as

electro-osmosis, chemical precipitation, ion exchange

or coagulation/flocculation can be used in the remedia-

tion of wastewaters containing heavy metal ions. Besides

being quite expensive, these methods are also strongly

biased by possible adverse chemical processes (White et

al., 1997). On the other hand, biosorption techniques

could represent a new, less expensive way to remove

toxic heavy metals even in dilute conditions from

industrial wastewaters (Gavrilescu, 2004).

e front matter r 2005 Elsevier Ltd. All rights reserve

atres.2005.04.022

ing author. Tel.: +39091 7028169;

4035.

ess: fsaiano@unipa.it (F. Saiano).

Among these techniques, those which use fungi have

been recently recognised as promising tools (Kapoor

and Viraraghavan, 1997; Say et al., 2001). In fact, the

purification of the water containing metals by fungal

biomass is cheaper and has the following advantages: (i)

production of small residual volume; (ii) possibility of

valorisation of fungal waste biomasses from industrial

fermentations; (iii) fast removal; (iv) easy installation of

the process.

Studies have focused on mycelium structure acting as

an adsorbing medium through its polysaccharidic

components (Bhanoori and Venkateswerlu, 2000;

Pinghe et al., 1999). On the other hand, it has been

reported that dried matter or, even better, biomaterials

obtained from alkali treated fungi, could provide higher

capacities than the whole fungus (Chandra Sekhar et al.,

d.

ARTICLE IN PRESSF. Saiano et al. / Water Research 39 (2005) 2273–22802274

1998; Kapoor et al., 1999; Alonzo et al., 2001). Such a

treatment usually yields a soluble fraction, mainly

composed of glycans, heteroglycans and glycoproteins,

and an insoluble material containing various amounts of

chitin/chitosan, cellulose and other b-glucans (Griffin,1994). On the other hand, increasing amounts of chitin

((b1-4)-N-acetyl-D-glucosamine) are hydrolysed to

chitosan, its deacetylated form, depending on the

strength of the alkali treatment (Muzzarelli et al.,

1980; Kurita, 2001).

It is interesting to note that the cell walls of different

fungi orders are able to give materials with various

associations of chitin/chitosan with glucans or other

polysaccharides, so that in principle a large variability in

biomaterial–pollutant interactions may be obtained. The

growing need for new sources of low-cost adsorbent

undoubtedly make chitosan-containing biomass one of

the most attractive materials for wastewater treatment

(Babel and Kurniawan, 2003).

Phomopsis sp. is an Ascomycetes filamentous fungus,

that has attracted our attention as a potential tool for

wastewater remediation because of its high radial

growth rate that can be produced using relatively

unsophisticated and low-cost culture propagation tech-

niques. Moreover, the main cell wall polysaccharides of

the Ascomycetes class are b-glucans and a-chitin(Griffin, 1994) in varying amounts depending on the

species.

This paper reports on the characterisation of the

polysaccharidic content of Phomopsis sp. cell wall and

on the evaluation of its complexing ability with a

number of metal aquo-ions.

Finally, the biomaterial has been tested against

authentic metal-ion containing urban wastewater.

2. Materials and methods

2.1. Chemicals

FT-IR grade potassium bromide, sodium hydroxide,

potassium peroxydisulfate and 37% hydrochloric acid

were reagent grade (Fluka). Chitin and chitosan from

crab shells were practical grade (Sigma). Sixty-five

percent nitric acid and 30% hydrogen peroxide were

Suprapur grade (Aldrich). Metal standards, ICP grade

solutions, were 1.00mg/mL in 5% Suprapur nitric acid

(Merck). Potato dextrose agar (DIFCO) and MilliQ

water (Millipore) were also used.

2.2. Fungal growth and biomaterial production

Standard cultures of Phomopsis sp. were obtained

from culture collections. Standard cultures were stored

on Potato–Dextrose–Agar (PDA) at 15 1C. For liquid

cultures, 9mm diameter disks taken from 10-day-old

cultures, grown on PDA at 23 1C in the dark, were used

to inoculate 1 L Roux bottles with 150mL of carrot

broth (the broth of 400 g carrots was strained through

cheesecloth and the volume made up to 1L with distilled

water). Cultures were further incubated for 10 days in

the dark. Mycelium was harvested by filtration and

washed several times with sterile water. Bottled-dry

mycelium was weighted and stored at �20 1C.

In a typical experiment, 100 g of frozen mycelium

were added in a beaker containing 0.5 L of a 0.5M

sodium hydroxide solution, and then boiled for 30min.

The resulting insoluble material was filtered, rinsed with

distilled water until neutrality and dried overnight in a

oven at 60 1C. The solid mass was carefully ground and

passed through a 100 mm sieve.

2.3. Characterization of the biomaterial

The determination of N and P content was carried out

by oxidation in a CEM MDS 2000 microwave oven

(ECO0009 method) (CEM, 1994). The amounts of

nitrate or phosphate ions produced were spectrophoto-

metrically determined (Spectroquant, Merck). Sulfur

(ECO0010 method) (CEM, 1994) and metal ions were

determined spectrophotometrically and by ICP-MS

4500 (Agilent), respectively.

2.4. Infrared measurements

Infrared spectra were recorded as KBr pellets in the

4000–400 cm�1 range on a Bruker Vector 22 FT-IR

instrument. The Bruker Opus 2 software allowed

sampling and manipulation of spectra.

2.5. Lewis sites determination

A suspension of 100mg of fungal biomaterial, in

100mL of 0.1mM HCl was stirred for 12 h. After this

time, the filtered solution was titrated with 10mM

NaOH solution. The difference of hydrogenion amounts

in solution gives the basic Lewis site equivalents in

biomaterial.

2.6. Batch kinetics adsorption

Solutions of 0.4mM containing single Cd, Cu, Ni, Pb

or Zn ions were obtained by diluting a corresponding

standard ICP grade. All the solutions were adjusted at

pH 6.070.1. Vials containing 100mL of metal ion

solution and 50mg of biomaterial were kept under

magnetic stirring. For each metal, a number of vials was

prepared so that, assuming a similar behaviour, the time

dependence of metal ion concentration could be

measured within 3 h. The filtered solutions were acidified

with HNO3 and 100mL of 0.4mM rhodium chloride

ARTICLE IN PRESSF. Saiano et al. / Water Research 39 (2005) 2273–2280 2275

were added as internal standard. Analyses were per-

formed by ICP-MS in quantitative mode.

2.7. Adsorption studies

Ten metal ion solutions were prepared as described

before at concentrations in the range 0.1–1mM at

constant pH values of 4.0, 5.0 or 6.0, respectively.

The selected range has been carefully chosen in order

to avoid hydrogen/cation competition against the

biomaterial as well as biomaterial/hydroxide ion com-

petition against the metal ions. Vials containing 10mL

of metal solution and 20mg of biomaterial were stirred

for 24 h. Afterwards, the suspension was filtered, and

100 mL of HNO3 were added to the filtrate before

analysis. The recovered biomaterial was contacted with

10mL of 0.25M nitric acid for 24 h. Subsequently, the

biomaterial was filtered again and the solution analysed.

Rhodium chloride of 0.4mM was added to each

sample as internal standard to avoid any instrumental

drift, and analysis was carried out by ICP-MS with

external calibration in quantitative mode.

2.8. Metal adsorption from wastewater

Adsorption experiments, as above, were performed

using wastewater obtained from the treatment plant of

Castelbuono (Palermo, Italy). Wastewater was pre-

viously filtered with a Millipore 0.45mm filter. The

COD (chemical oxygen demand) value of filtered

wastewater was found to be 51.6mg O2/L at pH ¼ 6.5.

Only one concentration of metal ions, 0.4mM, has been

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

121400160018002000

wavenumb

Abs

orba

nce

Fig. 1. Selected range of FT-IR spectra of chito

considered for each ionic species and pH was adjusted at

values of 4.0, 5.0 and 6.070.1, respectively. Theaddition of ionic standard solution to wastewater gave

rise to the precipitation of a metal containing flocculate.

After filtration the initial metal content of the solution

was determined. Twenty milligrams of biomaterial were

suspended in 100mL of the metal solution, the suspen-

sion stirred for 24 h, and the determination of metal

content both in the solution and in the biomaterial was

performed as described before.

3. Results and discussion

Preliminary characterisation of the biomaterial was

performed by the analysis of total nitrogen (5%),

phosphorus (1%), and sulfur (o0.1%). Metal ions ofinterest were below the detection limits. Considering

that nitrogen content is 9% in chitosan, the total

nitrogen value obtained points to a content of chitosan

around 60% in the whole biomaterial. Reasonably, the

remaining part consists mainly of glucans.

The FT-IR spectra of the biomaterial together with

those of commercial samples of chitin and chitosan, in

the range 2000–400 cm�1 are reported in Fig. 1. The

principal FT-IR features are given in Table 1.

The broad band in the range 3500–3000 cm�1 is

assigned to O–H and N–H stretching features of

hydroxylic and primary amine and amide groups. The

band at 1560 cm�1 present in the chitin spectrum and

assigned as Amide II is missing in the biomaterial and,

as expected, in the chitosan spectrum. On the above

400600800100000

er (cm-1)

chitosan

biomaterial

chitin

san, Phomopsis sp. biomaterial and chitin.

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Table 1

Wavenumbers, corresponding possible groups and characteristics of principal absorption bands of FT-IR spectra of biomaterial, chitin

and chitosan

IR banda Wavenumberb (cm�1)

Biomaterial Chitin Chitosan

nas,nsðN2HÞ and nas,ns(O–H) 3500–3000 vs, br 3450–3270 vs, br 3450–3270 vs, br

ns(C–H) 2920 s 2920m 2920m

nas(CQO) 1745m

Amide I 1650 s

d(N–H) amine 1647m 1645m

Amide II 1568w 1560 s

d(C–H) 1441m 1447m 1446m

n(C–OH) 1156 vs 1195 s 1200 s

aMovement type: nas, asymmetric stretching; ns, symmetric stretching: d, bending.bBand intensity: vs, very strong; s, strong; m, medium; w, weak; sh, shoulder; br, broad.

F. Saiano et al. / Water Research 39 (2005) 2273–22802276

evidence, the band present in all the spectra at about

1650 cm�1 is confidently assigned as amide I in the chitin

spectrum and, instead, as primary amine both in the

biomaterial and in the chitosan spectra. The intensity

ratio between the features at 1650 and 1200 cm�1 is

strongly evident in biomaterial spectra when compared

with chitin and chitosan spectra. This evidence is in

agreement with the analytical data reported above.

Information on the chitin/chitosan molar ratio can be

obtained using the following equation (Kim et al., 1997):

DDð%Þ ¼ ½1� ðA1655=A3450Þ=1:33� � 100, (1)

where DD correspond to the deacetylation degree, A1655

and A3450 to the absorbance values recorded at 1655 and

3450 cm�1, characteristic for carbonyl and hydroxyl

bands, respectively. The application of the equation

gives a value of 92% of DD in the biomaterial.

Lewis basic sites were 340, 180 and 480mmol/g forbiomaterial, chitin and chitosan respectively. Consider-

ing the different basicity shown by the acetamide groups

present in chitin in comparison with the amino groups in

chitosan, above data accounts for the different values

observed for these two matrices (Zhang et al., 1998).

Moreover, the intermediate value of the biomaterial

confirms, the analytical nitrogen data and the relevant

contribution of chitosan respect to chitin as seen by FT-

IR.

3.1. Metal ion adsorption and isotherm models

The results of the adsorption studies are reported in

Fig. 2 representing the amount of heavy metal adsorbed

by the biomaterial at the higher concentration tested, for

each pH value chosen. Recovery tests from metal

adsorbed biomaterial indicate a 9872% efficiency.

Table 2 shows some of the adsorption capacities

reported in the literature and chitosan is reported as a

reference. Sorption depends heavily on experimental

conditions such as pH, metal concentration, ligand

concentration, competing ions and particle size. Un-

fortunately, some confusion persists in the published

literature when it comes to quantitatively expressing and

evaluating biosorption performance. The quantitative

foundation for comparing any sorption process is in the

relatively simple batch equilibrium contact experiment.

It allows enough time for establishing equilibrium

between the metal immobilized, sequestered in the solid

material (sorbent) and the metal still left in the solution.

Although our biomaterial, mainly for lead, cadmium,

copper and zinc, appears to compete favourably with

other biomaterials, it should be stressed that the

experimental conditions involved in the literature are

heterogeneous and great care has to be taken when

comparing these data.

Adsorption data for the different metal ion species are

related to their respective affinities for the biomaterial,

showing mainly amino and hydroxy functional groups.

As expected, nickel, zinc and cadmium are found to be

adsorbed to a lesser extent than lead and copper ions,

according to the literature data on their formation of

complexes with ligands bearing amino- and hydroxy-

groups (Harvey, 2000; Merce et al., 2001).

Assuming that the maximum number of complexing

sites corresponds to the determined Lewis basic equiva-

lents, its seems possible to argue, from data in Fig. 2,

that some ligand sites still remain uncomplexed. On the

other hand, when lead and copper are at the highest

concentration, adsorptions up to 2.5 and 5 times,

respectively, were observed.

To explain the data reported in Fig. 2, we have

evaluated some possible isotherm adsorption models.

The interpretation of collected data is in principle done

using Langmuir and Freundlich isotherm models which

assume a monolayer solute adsorption. The Freundlich

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Table 2

Literature reported adsorption capacities (mg/g dry weight) of different polysaccharides-containing biomaterials

Source of material Relative mass (mg/g dry weight) Material used Reference

Cd2+ Cu2+ Ni2+ Pb2+ Zn2+

Phomopsis sp. 26 25 6 179 10 Treated biomass Present work

Auricularia polytricha 1.01 Mycelium Galli et al. (2003)

Rhizopus arrhizus 18.6 104 Biomass Selatnia et al. (2004a (Ref. 1,3,50))

Streptomyces rimosus 32.6 135 Treated biomass Selatnia et al. (2004b)

Pseudomonas aeruginosa 265 Biomass Selatnia et al. (2004a)

Zoogloea ramigera 54 Biomass Selatnia et al. (2004a)

Zostera noltii 48.3 Biomass Sebe et al. (2004)

Penicillium chrysogenum 116 Biomass Selatnia et al. (2004a (Ref. 49))

Bacillus lentus 30 30 15 Treated biomass Vianna et al. (2000)

Saccharomyces cerevisiae o5 o5 5 Treated biomass Vianna et al. (2000)

Aspergillus oryzae 30 13 12 Treated biomass Vianna et al. (2000)

Chlorella minutissima 11.14 9.74 Treated biomass Roy et al. (1993)

Streptomyces griseus 28 Treated biomass Matis and Zouboulis (1994)

Chitosan 5.93 222 164 16.36 75 Pure substance Babel and Kurniawan (2003)

0

100

200

300

400

500

600

700

800

900

Pb Cu Cd Zn Ni

Rel

ativ

e ad

sorb

ed a

mou

nt (

mic

rom

ol /

g ad

sorb

ent)

pH 4 pH 5 pH 6

Fig. 2. Amounts of metal ions adsorbed by 20mg of biomaterial stirred for 24 h with 10mL of a 1.0mmol/L solution of each metal

tested at the three pH values.

F. Saiano et al. / Water Research 39 (2005) 2273–2280 2277

model is empirically correlated with the logarithm of the

heat of adsorption, decreasing with the fraction of

surface covered by the solute. The Langmuir model

refers only to the solution concentration and to the

available adsorption surface (Ozer et al., 1999; Sag et al.,

1998). Moreover, a further development of the Lang-

muir model is the BET isotherm that describes an

interaction of a solute with an adsorbent matrix in which

as the metal ion concentration increases, a distribution

of the solute in multiple layers follows the covering of all

the exposed functional sites.

Langmuir model:

qeq ¼ Q0BCeq=ð1þ BCeqÞ, (2)

where qeq is the equilibrium adsorbate loading on the

adsorbent, Ceq the equilibrium concentration of the

adsorbate, Q0 the ultimate capacity, B the relative

energy (intensity) of adsorption, also known as binding

constant.

The Langmuir model was linearised to obtain the

parameters Q0 and b from experimental data by plotting

ARTICLE IN PRESSF. Saiano et al. / Water Research 39 (2005) 2273–22802278

the equilibrium concentrations versus the adsorbent

loading.

Freundlich model:

Qeq ¼ K fCneq, (3)

where Qeq is the ion concentration in adsorbent

material, Ceq the ion concentration in aqueous phase

at equilibrium time, K f the Freundlich adsorption

constant, n is the Freundlich exponent.

BET model:

Qeq ¼ QmBCeq=ðCs � CeqÞ½1þ ðB � 1ÞðCeq=CsÞ�. (4)

And the linearised shape is (5)

ðCeq=CsÞ=Qeqð1� Ceq=CsÞ

¼ 1=QmB þ ðB � 1=QmBÞðCeq=CsÞ, ð5Þ

where Ceq is the amount of ions in solution after the

equilibrium time, Cs the initial amount of ions used in

the experimental approach, Qm the amount of ions that

cover the layer of bioadsorbent, Qeq the amount of ions

trapped by the system, B is the affinity constant between

the ion species and the bioadsorbent material.

Data collected for cadmium, zinc and nickel fit both

Langmuir and Freundlich models, mainly confirming

the presence of monolayers.

It is interesting to note that the larger adsorbed

amounts of lead and copper would suggest the possible

presence of multilayer also due to the good affinity of

the metal ions for the adsorbent material. Therefore the

monolayer-based Langmuir model would not be in these

cases quite reasonable.

Table 3

Sorption isotherm coefficients of Langmuir, Freundlich and BET mo

Metal pH Langmuir parameters Freundlic

Q0 (mmol/g) B (mL/g) R2 Kf

Cu 4.0 330780 13276 0.77 917185.0 270780 11074 0.63 667146.0 290770 140710 0.58 70715

Pb 4.0 490720 71.570.8 0.97 1267185.0 570740 95.570.3 0.96 1507206.0 680770 5671 0.95 111719

Cd 4.0 250730 9.670.2 0.89 17735.0 19678 13.170.6 0.95 31756.0 260710 10.670.1 0.98 2876

Ni 4.0 10273 19.570.4 0.98 18745.0 9676 3272 0.88 20746.0 10374 3071 0.95 2073

Zn 4.0 11574 20.170.6 0.97 19745.0 15774 14.070.1 0.99 20746.0 13579 3072 0.89 33711

While the data for copper agree well with the BET

model only, for lead, even though data appears to fit

both models, we still choose the model accounting for

the multilayer arrangement.

The parameters obtained by linearising the equations

are shown in Table 3.

3.2. Batch kinetic experiments

Batch kinetics experiments are given in Fig. 3 showing

the concentration decrease of the various metal ion

species in the first 30min.

Decrease in the metal ion concentration was reason-

ably fast during the first 10–15min in which biomaterial

appeared covered for about 70% of the functional sites

exposed. Equilibrium condition was achieved within 3 h.

3.3. Adsorption in wastewater media

In Table 4, the results obtained by testing single metal

ion sorption at the three pH values are shown. In this

medium, the adsorbed relative amounts are similar to

those recorded in distilled water. The best results were

obtained for copper and lead which underwent a very

good elimination by the biomass, whereas lower

efficiency levels were found for nickel, zinc and cadmium

(36%, 22% and 20%, respectively). Generally, the

highest adsorption capacity was found at the highest

pH value.

When compared to recovery reported for other

commercialised metal biosorbents, our data are en-

dels

h parameters BET parameters

n R2 Qm (mmol/g) B R2

0.2270.04 0.76 180715 160730 0.95

0.2670.05 0.80 170712 97712 0.95

0.2770.05 0.80 220720 27710 0.95

0.2370.03 0.88 199712 2572 0.96

0.2470.05 0.87 250720 3673 0.96

0.3370.04 0.90 209716 1572 0.87

0.4070.03 0.95

0.2770.03 0.93

0.3270.04 0.90

0.2770.04 0.87

0.2570.04 0.85

0.2670.05 0.92

0.2870.04 0.87

0.3070.03 0.91

0.2170.06 0.63

ARTICLE IN PRESS

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0 5 10 15 20 25 30time (min)

met

al io

n co

ncen

trat

ion

( m

mol

/ L)

NiCuZnPbCd

Fig. 3. The adsorption extent of 50mg of biomaterial put in contact with 0.4mmol/L ions solution (pH ¼ 6.0) within 30min

experiment.

Table 4

Recovery efficiency and recovered amounts from 20mg of biomaterial contacted in 100mL of the tested metal ions in wastewater

stirred for 24 h at three pH values

Metal pH Initial amount in

the sample (mmol)Recovered amount in

the biomaterial (mmol)Relative recovered amount

(mmol/g adsorbent)Recovery efficiency

(%)

Cd 4.0 2.5 0.24 12 9.6

5.0 2.4 0.33 16 14

6.0 2.5 0.51 26 20

Cu 4.0 3.6 0.92 46 26

5.0 3.5 0.90 45 26

6.0 1.4 1.4 68 100

Ni 4.0 3.6 0.27 14 7.5

5.0 3.7 0.71 36 19

6.0 2.8 1.0 51 36

Pb 4.0 0.66 0.40 20 61

5.0 0.61 0.53 26 87

6.0 0.67 0.47 23 70

Zn 4.0 3.1 0.41 21 13

5.0 3.1 0.64 32 21

6.0 3.1 0.67 33 22

Standard deviations of the data lie in the range 10–15%.

F. Saiano et al. / Water Research 39 (2005) 2273–2280 2279

couraging (Gavrilescu, 2004; Coulibaly et al., 2003;

Bailey et al., 1999; Babel and Kurniawan, 2003).

4. Conclusions

The characterisation of the biomaterial suggests a

relevant contribution from a glucans–chitosan mixture.

This material shows the ability to complex various metal

species from aqueous media, mainly due to the presence of

chitosan. Adsorption as a monolayer, which is found to

follow Langmuir and Freundlich models, holds for most

metal ions except for copper and lead, for whose it is better

described by the BET multilayer distribution model.

Both in distilled water and in urban wastewater, the

studied metal ions are adsorbed following the sequence

ARTICLE IN PRESSF. Saiano et al. / Water Research 39 (2005) 2273–22802280

lead4copper4zincXcadmiumXnickel. This trend does

not seem to be significantly influenced by the acidity of

the medium. The results obtained during this study show

that this method of removing heavy metal ions is

promising compared to other conventional and gener-

ally more expensive processes.

Other advantages are a relatively low growing-cost for

a large amount of biomass, a cheap procedure to obtain

the treated biomaterial and a not detrimental process for

recovery of surface-bound metals.

Extension of the present study to other fungi orders is

in progress with the aim of increasing our knowledge on

the cell wall matter and its ability to complex metal ions,

as well as its possible applications in bioremediation

uses.

Acknowledgements

Funds from MIUR ‘‘Ministero dell’Istruzione dell’U-

niversita e della Ricerca’’ are acknowledged.

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