ARTICLE IN PRESS

0043-1354/$ - se

doi:10.1016/j.w

�Correspondfax: +9140 716

E-mail add

(K.C. Sekhar).1Present add

Metallurgical R

500 058, India.

Water Research 39 (2005) 2815–2826

www.elsevier.com/locate/watres

Removal of arsenic(III) from aqueous solutions using freshand immobilized plant biomass

C.T. Kamalaa, K.H. Chub, N.S. Charya, P.K. Pandeyc, S.L. Rameshd,A.R.K. Sastrye, K.Chandra Sekhara,�,1

aAnalytical Chemistry and Environmental Sciences Division, Indian Institute of Chemical Technology, Uppal Road, Habsignda,

Hyderabad 500 007, AP, IndiabDepartment of Chemical and Process Engineering, University of Canterbury, Private Bag 4800, Christchurch, New Zealand

cCenter for Environmental Science & Engineering, Department of Engineering Chemistry, Bhilai Institute of Technology, Durg, CG, IndiadGeochemistry Division, National Geophysical Research Institute, Hyderabad 500 007, AP, India

eOrganic Coatings and Polymers Division, Indian Institute of Chemical Technology, Hyderabad 500 007, AP, India

Received 23 June 2004; received in revised form 28 March 2005; accepted 26 April 2005

Available online 5 July 2005

Abstract

The ability of Garcinia cambogia, an indigenous plant found in many parts of India, to remove trivalent arsenic from

solution was assessed. Batch experiments were carried out to characterize the As(III) removal capability of fresh and

immobilized biomass of G. cambogia. It was found that the kinetic property and uptake capacity of fresh biomass were

significantly enhanced by the immobilization procedure. The uptake of As(III) by fresh and immobilized biomass was

not greatly affected by solution pH with optimal biosorption occurring at around pH 6–8. The presence of common

ions such as Ca and Mg at concentrations up to 100mg/l had no effect on As(III) removal. However, the presence of

Fe(III) at 100mg/l caused a noticeable drop in the extent of As(III) removal but the effect was minimal when Fe(III)

was present at 10mg/l. The adsorption isotherms quantitatively predicted the extent of As(III) removal in groundwater

samples collected from an arsenic-contaminated site in India. Immobilized biomass loaded with As(III) was amenable

to efficient regeneration with NaOH solution. Column studies showed that immobilized biomass could be reused over

five cycles of loading and elution. The excellent As(III) sequestering capability of fresh and immobilized G. cambogia

biomass could lead to the development of a viable and cost-effective technology for arsenic removal in groundwater.

r 2005 Elsevier Ltd. All rights reserved.

Keywords: Adsorption; Arsenic; Biosorption; Column; Immobilization; Plant biomass

e front matter r 2005 Elsevier Ltd. All rights reserve

atres.2005.04.059

ing author. Tel.:+91 40 27153884;

0387.

resses: kavi@iict.res.in, kavi223@yahoo.com

ress: Analytical Chemistry Group, Defense

esearch Laboratory, Kanchanbagh, Hyderabad

1. Introduction

Arsenic contamination in drinking water and ground-

water has created serious health problems in countries

like India and Bangladesh and in other parts of the

world (Das et al., 1996; Pandey et al., 2002; Chandra

Sekhar et al., 2003a; Wang and Wai, 2004). As a result,

extensive research has been focused on develop-

ing arsenic removal methods in recent times. The

d.

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Nomenclature

ARE absolute relative error

b Langmuir binding constant (l/mg)

Ce As(III) concentration of solution phase at

equilibrium (mg/l)

Ci initial As(III) concentration of solution

phase (mg/l)

IGCFIX immobilized Garcinia cambogia biomass

PR As(III) percent removal

PRexp experimental As(III) percent removal

PRcal calculated As(III) percent removal

qe As(III) concentration in the adsorbent phase

at equilibrium (mg/g)

qm maximum uptake capacity of the adsorbent

(mg/g)

w adsorbent dosage (g/l)

C.T. Kamala et al. / Water Research 39 (2005) 2815–28262816

conventional arsenic removal method is precipitation–

coagulation with lime and Fe(III) salts followed by

adsorption onto the resulting Fe(III) hydroxide flocs. A

problem with this technique is the separation and

handling of the hazardous sludge. Recently, adsorption

processes have attracted considerable attention owing to

their ability to treat aqueous solutions contaminated

with trace quantities of arsenic. Various adsorbent

materials have been tested for their ability to remove

the two inorganic arsenic species commonly found in

water; arsenite [As(III)] (Singh and Pant, 2004; Thir-

unavukkarasu et al., 2005) and arsenate [As(V)] (Xu et

al., 2002; Dousova et al., 2003; Rau et al., 2003; Zhang

et al., 2003; Genc--Fuhrman et al., 2004a; Vaishya and

Gupta, 2004). Simultaneous removal of both arsenic

species by adsorption has also been reported (Elizalde-

Gonzalez et al., 2001; Chakravarty et al., 2002;

Katsoyiannis and Zouboulis, 2002; DeMarco et al.,

2003; Zeng, 2003; Daus et al., 2004; Genc--Fuhrman et

al., 2004b; Kim et al., 2004). In general, As(III), which

exists predominantly as nonionic H3AsO3 in natural

waters, is more difficult to remove compared to As(V),

which exists predominantly as deprotonated oxyanions

H2AsO�4 or HAsO2�

4

� �(DeMarco et al., 2003). As a

result, some of the proposed arsenic removal methods

require a preoxidation step which oxidizes As(III) to

As(V) (Bissen and Frimmel, 2003). A literature review

on adsorption processes based on inorganic adsorbents

for arsenic removal is available elsewhere (Dambies,

2004).

Numerous biological materials have been tested for

removal of toxic ions from aqueous solutions over the

last two decades. However, only a limited number of

studies have investigated the use of adsorbents derived

from biological sources, e.g., chitosan (Mcafee et al.,

2001; Dambies et al., 2002), alginate (Zouboulis and

Katsoyiannis, 2002), orange waste (Ghimire et al., 2002,

2003), fungal biomass (Loukidou et al., 2003; Say et al.,

2003a, b), methylated yeast biomass (Seki et al., 2005),

and chicken feathers (Teixeira and Ciminelli, 2005), to

remove arsenic from aqueous solutions. Biological

materials represent a potential source of abundant,

low-cost adsorbents. This communication investigates

biosorption of As(III) by Garcinia cambogia, a yellowish

pumpkin shaped tropical tree fruit native to India.

Previous studies have demonstrated the effectiveness of

similar plant biomass in removing toxic ions from

aqueous solutions (Chandra Sekhar et al., 2003b, 2004).

Because most biological materials are fragile and have a

tendency to swell when wet, they are not suitable for use

in standard process equipment such as continuous-flow

column contactors which are often operated under high

pressure conditions. However, if the biomass is chemi-

cally reinforced or immobilized on a solid support, the

resulting product becomes useful for applications in

column contactors without causing operational pro-

blems such as clogging and pressure drop fluctuations.

One of the earliest examples of immobilized biomass is

the polymeric beads containing sphagnum peat moss or

algal biomass developed by the US Bureau of Mines.

The immobilized biomass exhibited excellent adsorption

capability for toxic ions including arsenic (Jeffers et al.,

1991). In this study, batch experiments were performed

to evaluate the adsorption characteristics of fresh and

immobilized G. cambogia biomass for As(III) in

synthetic solutions as well as actual groundwater

samples. Previous studies reported that the groundwater

samples examined in the current study contained

relatively high concentrations of As(III) (Pandey et al.,

2002; Chandra Sekhar et al., 2003a). Continuous-flow

experiments were then performed using a column

packed with immobilized biomass to evaluate As(III)

removal and recovery over multiple cycles of operation.

2. Materials and methods

2.1. Preparation of immobilized biomass

Immobilized G. cambogia biomass beads, designated

as IGCFIX, were prepared using a method developed by

Indian Institute of Chemical Technology. The dried fruit

rinds of G. cambogia were used. A wide range of bead

fabrication parameters were tested to identify factors

having the most significant effects on the chemical and

physical characteristics of immobilized biomass beads

(Jeffers et al., 1991). Variables investigated included the

concentration of biomass in bead, the solvent type, the

ARTICLE IN PRESSC.T. Kamala et al. / Water Research 39 (2005) 2815–2826 2817

composition and temperature of the polymerizing

media, and the bead curing time. The amount of raw

biomass in an optimized bead constituted 0.95 g per

gram of total bead weight. The polymerization was

performed at 2972 1C. Water was found to be the best

solvent and the bead curing time was set at 30min which

allowed biomass to become tack free. The average bead

size was about 1mm.

2.2. Batch studies

As(III) stock solutions were prepared by dissolving

NaAsO2 salt in deionized water. A known amount of

fresh biomass or IGCFIX was washed twice with 0.01M

HCl to remove any debris or soluble components that

might interact with As(III). Batch kinetic studies were

first conducted with fresh biomass or IGCFIX to

determine the time needed for the As(III) binding

process to reach the equilibrium state. Based on the

kinetic experiment results, all equilibrium experiments

were conducted for a period of 30min. Flasks contain-

ing fresh biomass or IGCFIX in 100ml As(III) solutions

were agitated in a mechanical shaker (Techno India Ltd)

at 300 rpm and 30 1C. Upon equilibration solution

samples were filtered and analyzed by GF-AAS for

As(III). The effects of process variables such as pH

(2–10), biomass dosage (5–40 g/l), initial As(III) con-

centration (50–2500mg/l), and background ions (Ca,

Mg, and Fe(III)) on As(III) uptake were investigated. In

addition, batch elution experiments were carried out to

desorb bound As(III) from IGCFIX using different

eluting agents such as HCl, HNO3, NaCl, Na2CO3, and

NaOH. Finally, water samples collected from the

groundwater of West Bengal and Chhathisgarh which

is believed to be contaminated with arsenic released by

natural processes and from the groundwater of Patan-

cheru which is believed to be contaminated with arsenic

discharged by industrial sources were treated with fresh

biomass or IGCFIX in batch experiments. The sam-

pling, preservation, and storage of groundwater samples

were carried out in accordance with the prescribed

methods which have been described in detail elsewhere

(Pandey et al., 2004).

2.3. Column studies

Experiments using a laboratory-scale column were

conducted to investigate As(III) removal by IGCFIX

under continuous-flow conditions, elution of the bound

As(III), and regeneration of IGCFIX for reuse. IGCFIX

beads were packed into a column having an internal

diameter of 1.6 cm and a column height of 20 cm.The

active height of the packed bed was 7 cm. An aqueous

solution containing 100mg/l As(III) was passed through

the column at a flow rate of 13.3ml/min. The pH of the

feed solution was continuously monitored and main-

tained constant by addition of 0.01M NH4OH or HCl.

Elution of the bound As(III) from IGCFIX was carried

out using an aqueous 0.05M NaOH solution at a flow

rate of 1ml/min. Eluted IGCFIX beads were washed

with deionized water for regeneration. Regenerated

IGCFIX beads were tested for As(III) uptake to

determine their reusability. Five cycles of As(III) loading

and elution were conducted.

2.4. Spectroscopic studies

In order to investigate the main functional groups

responsible for As(III) adsorption and the chemical

nature of the adsorbed arsenic, Fourier transform

infrared (FT-IR) and electron spectroscopy for chemical

analysis (ESCA) spectra of fresh biomass and IGCFIX

samples were obtained. Fresh biomass or IGCFIX was

first loaded with As(III) by contacting 1 g of biomass

with 100ml of a 1000mg/l As(III) solution at pH 6 for

30min. The As(III)-laden biomass was then collected by

filtration, washed with distilled water, and finally dried

in an oven. Additional fresh biomass and IGCFIX

beads were treated the same way except for the absence

of As(III). Infrared spectra of biomass samples were

recorded on a Nexus 670 FT-IR spectrometer (Thermo

Nicolet, USA). Disks of 150mg KBr containing 5mg of

finely ground powder of each sample were prepared for

the FT-IR analysis. ESCA spectra were acquired on a

Kratos/Shimadsu AXIS 165 system (Kratos Analytical,

UK). The X-ray source was Mg Ka radiation

(1253.6 eV) operated at 75W and 5mA. The instrument

was operated with a pass energy of 40 eV for high-

resolution scans.

3. Results and discussion

3.1. Spectroscopic observations

To better understand the involvement of main

functional groups in As(III) binding and the chemical

nature of the adsorbed arsenic, FT-IR and ESCA

spectra of fresh biomass and IGCFIX samples were

obtained. The resulting infrared spectra of fresh biomass

and IGCFIX gave similar results. Thus, only the spectra

of fresh biomass before and after As(III) adsorption are

shown in Fig. 1. The band assignments are given in

Table 1. As seen in Fig. 1, both spectra show the O–H

stretching region from 4000 to 2000 cm�1 and the

fingerprint region from 2000 to 1200 cm�1. For pristine

biomass the band at 1626 cm�1 can be ascribed to the

asymmetrical C¼O stretching of the carboxyl group

while the band at 1737 cm�1 is probably due to the

vibration of C¼O of the ester group. The latter C¼O

band at 1737 cm�1 was invisible on the spectrum of

As(III)-loaded biomass. Another noticeable change is

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Table 1

Assignments of infrared absorption bands for pristine and

As(III)-loaded biomass samples

Wavenumber (cm�1) Assignment

Pristine biomass As(III)-loaded biomass

3427 3417 O–H stretching

1737 – C¼O stretching

1626 1602 C¼O stretching

– 1429 C¼O stretching

Band assignments were made according to Lewis and Neela-

kantan (1965).

3417

.32

2922

.65

1602

.88

1429

.85

1265

.33

1104

.39

803.

90 603.

85

483.

2652

3.91

1066

.32

1212

.20

1626

.85

1737

.88

2925

.69

3427

.48

80

86

92

98

75

85

95

80

90

5001000150020003000

3417

.32

2922

.65

1602

.88

1429

.85

1265

.33

1104

.39

803.

90 603.

85

483.

2652

3.91

1066

.32

1212

.20

1626

.85

1737

.88

2925

.69

3427

.48

% T

rans

mitt

ance

% T

rans

mitt

ance

(a)

(b) Wavenumber (cm-1)

Fig 1. FT-IR spectra of (a) fresh biomass of G. cambogia and (b) As(III)-loaded biomass of G. cambogia.

C.T. Kamala et al. / Water Research 39 (2005) 2815–28262818

that the C¼O vibration band at 1626 cm�1 observed for

the spectrum of pristine biomass shifted to 1602 cm�1

with a different magnitude for As(III)-loaded biomass.

Furthermore, the spectrum of As(III)-loaded biomass

displayed a peak at 1429 cm�1 which can be ascribed to

the symmetrical C¼O stretching of the carboxyl group.

These band changes indicate the carboxyl group

involvement in As(III) adsorption, probably through

chelation/complexation. Carboxyl group is a character-

istic group of hydroxycitric acid which is the principal

organic acid found in G. cambogia (Jena et al., 2002).

Hydroxycitric acid has a chemical structure similar to

that of citric acid. It has been shown that As(III) was

able to reduce citric acid content in citrus fruit although

the exact interaction mechanism has not been elucidated

(Sadka et al., 2000). It is postulated that the carboxyl

group of hydroxycitric acid was involved in As(III)

binding through chelation/complexation. ESCA is a

spectroscopic technique for chemical analysis of surfaces

and can provide information about the oxidation states

of an element. The overall ESCA spectrum for a sample

of As(III)-loaded biomass is shown in Fig. 2a. An

enlargement of the As(3d) binding energy region from

Fig. 2a is shown in Fig. 2b. It is evident that the As(3d)

peak position for the As(III)-loaded biomass sample

strongly correlates with the presence of an As(III)

surface species.

3.2. Batch studies

3.2.1. Kinetics of As(III) removal

Batch kinetic experiments were performed to deter-

mine the time required for the As(III) binding process to

reach equilibrium. Fig. 3 shows time profiles for As(III)

removal by the two types of biomass in batch systems

maintained at pH 6 and containing an aqueous 100mg/l

As(III) solution and 5 g/l biosorbent. It can be seen that

As(III) binding by fresh biomass reached equilibrium

after a contact time of �30min, yielding a maximum

removal of 90%. In contrast, equilibrium was reached

within 5min of contact in the case of IGCFIX. A

removal level of �100% was observed which remained

stable up to a contact time of 60min, after which it

decreased marginally following another 60min of

contact. The rapid binding of As(III) by IGCFIX

suggests that most of the As(III) binding sites of G.

cambogia cell walls are located on the exterior surfaces

of the beads, thereby eliminating intraparticle diffusion

which is a relatively slow rate process. The slower

binding kinetics exhibited by fresh biomass implies that

intracellular diffusion within the cell wall structure could

be a rate limiting step. The cell wall structure was

presumably destroyed by the immobilization procedure,

enabling immobilized biomass to exhibit faster uptake

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Fig. 2. (a) ESCA spectrum of As(III)-loaded biomass of G.

cambogia. (b) An enlargement of the As(3d) binding energy

region.

50

60

70

80

90

100

0 20 40 60 80 100 120 140Time (min)

As(

III)

perc

ent

rem

oval

Fresh biomassImmobilized biomass

Fig. 3. Time profiles for As(III) removal by fresh and

immobilized biomass.

50

60

70

80

90

100

0 42 6 8 10pH

As(

III)

perc

ent r

emov

al

Fresh biomassImmobilized biomass

Fig. 4. Effect of pH on As(III) removal by fresh and

immobilized biomass.

C.T. Kamala et al. / Water Research 39 (2005) 2815–2826 2819

kinetics. Based on the kinetic results presented in Fig. 3,

a contact time of 30min was selected for all subsequent

batch uptake experiments with either fresh biomass or

IGCFIX in order to ensure that equilibrium was

established in each case.

3.2.2. Effect of pH

Experimental results showing As(III) removal at

varying pH (2–10) are presented in Fig. 4. The figure

clearly shows that both types of biomass exhibited

maximum As(III) removal from batch systems contain-

ing an aqueous 100mg/l As(III) solution and 5 g/1

biosorbent within the pH range of 6–8. As(III) removal

was insensitive to pH variations within this range. This

finding indicates that fresh biomass or IGCFIX would

perform optimally in treating As(III)-contaminated

drinking water or groundwater whose pH values

typically fall within the same range. The relative

distribution of dissolved arsenic species (As(III) and

As(V)) is influenced by pH and the redox condition.

Under mildly reducing conditions and at pH below 9,

As(III) exists predominantly as H3AsO3 (DeMarco et

al., 2003). It may be inferred that G. cambogia biomass is

capable of binding nonionic H3AsO3 within the typical

pH range of natural waters (pH 6–8).

3.2.3. Effect of biosorbent dosage

The extent of As(III) removal as a function of fresh

biomass or IGCFIX dosage (5–40 g/l) in batch systems

maintained at pH 6 and containing an aqueous 100mg/l

As(III) solution was studied. The results are presented in

Fig. 5. The percent removal of As(III) increased from 90

to �100% when the dosage of fresh biomass was

increased from 5 to 40 g/l.When the initial As(III)

concentration and the volume of solution are fixed, the

fraction of As(III) removed from a batch contactor will

increase with increasing biosorbent dosage. The trend

displayed by fresh biomass is therefore expected when

the percent removal of As(III) is less than 100%. The

percent removal of a given biosorbent dosage may be

predicted from the adsorption equilibrium isotherm, as

will be discussed later. An increasing trend of percent

removal was not observed in the case of IGCFIX

because the lowest dosage of IGCFIX tested (5 g/l) was

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50

60

70

80

90

100

0 500 1000 1500 2000 2500

As(

III)

perc

ent

rem

oval

.

Fresh biomassImmobilized biomass

Initial As(III) concentration (mg/l)

Fig. 6. Effect of initial concentration on As(III) removal by

fresh and immobilized biomass.

50

60

70

80

90

100

0 10 20 30 40

As(

III)

perc

ent

rem

oval

Fresh biomassImmobilized biomass

Biosorbent dosage (g/l)

Fig. 5. Effect of biosorbent dosage on As(III) removal by fresh

and immobilized biomass.

C.T. Kamala et al. / Water Research 39 (2005) 2815–28262820

able to remove virtually all the As(III) present in the

batch system. It is obvious that further increase in

IGCFIX dosage will have no effect on the percent

removal of As(III). No noticeable As(III) removal

was observed in similar experiments using beads

made from the immobilization material only, indicating

that As(III) uptake can be attributed entirely to G.

cambogia biomass contained within immobilized bio-

mass beads. The practically important conclusion from

Fig. 5 is that immobilized biomass can easily outperform

fresh biomass, requiring a much lower biosorbent

dosage to achieve quantitative removal of As(III). The

immobilization procedure has not resulted in any

adverse effects on the As(III) uptake capability of G.

cambogia biomass. On the contrary, it appears to

enhance As(III) binding, presumably by exposing

previously inaccessible binding sites in fresh biomass

to As(III) or creating new binding sites having an

affinity for As(III).

3.2.4. Effect of initial As(III) concentration

The effect of initial As(III) concentration on the

extent of removal is shown in Fig. 6. The biosorbent

dosage and pH for these batch experiments were fixed at

5 g/l and pH 6. As expected, increasing the initial As(III)

concentration while keeping the other conditions con-

stant in a batch system resulted in decreasing percent

removal. In the experiments with fresh biomass percent

removal dropped rapidly from about 95 to 58% when

the initial As(III) concentration was increased from 50

to 1000mg/l. In contrast, IGCFIX exhibited a relatively

slow reduction in percent removal over a much larger

initial As(III) concentration range; at the maximum

initial As(III) concentration tested (2500mg/l), the

percent removal of As(III) remained at about 92%.

The results in Fig. 6 confirm that the As(III) uptake

capacity of IGCFIX is significantly larger than that of

fresh biomass.

3.2.5. Adsorption isotherms

The observed effects of biosorbent dosage and initial

As(III) concentration described above indicate that

IGCFIX can easily outperform fresh biomass with

regard to As(III) removal. A more systematic and

quantitative way of judging and comparing adsorbent

performance is through adsorption isotherm analysis.

When equilibrium is established in a batch adsorption

system, the adsorbate concentration in the adsorbent

phase is given by the following mass balance equation:

qe ¼ qi þCi � Ceð Þ

w, (1)

where qe (mg/g) and Ce (mg/l) are the adsorbate

concentration in the adsorbent and solution phase at

equilibrium, qi (mg/g) and Ci (mg/l) are the initial

adsorbate concentration in the adsorbent and solution

phase, and w (g/l) is the adsorbent dosage. Experimen-

tally generated qe vs. Ce equilibrium data are used to

construct the adsorption isotherm graphically which can

be modeled in terms of isotherm models such as the

simple Langmuir and Freundlich equations.

For liquid-phase adsorption systems containing a

single adsorbate, the adsorption isotherm is a function

of environmental factors such as pH, ionic strength, and

temperature. Once these factors are fixed, the adsorption

isotherm is, in principle, independent of the experi-

mental conditions employed to measure it. In other

words, a unique isotherm can be generated by using any

convenient measurement method (batch or continuous-

flow) and by varying either w or Ci. Adsorption

isotherms for As (III) on fresh biomass and IGCFIX

may thus be constructed from the equilibrium data

shown in Figs. 5 and 6 which have been generated by

varying w and Ci. In the case of IGCFIX, adsorption

isotherm cannot be constructed from the equilibrium

data of Fig. 5 because the percent removal of As(III)

was �100% in all cases. On the other hand, the

ARTICLE IN PRESSC.T. Kamala et al. / Water Research 39 (2005) 2815–2826 2821

equilibrium data of Fig. 6 indicate that appreciable

amounts of As(III) were still present in solution at

equilibrium (percent removalo100%). For fresh bio-

mass, adsorption isotherm can be constructed from the

equilibrium data of either Fig. 5 or Fig. 6 because the

percent removal of As(III) in most cases was o100%. It

was decided to use the equilibrium data of Fig. 6 to

construct adsorption isotherms for As(III) on the two

types of biomass.

The percent removal of As(III) (PR) is given by

PR ¼ 100Ci � Ce

Ci

� �. (2)

Combining Eqs. (1) and (2) yields

qe ¼Ci

w

PR

100. (3)

Values of qe estimated from the percent removal data of

Fig. 6 using Eq. (3) are plotted against the correspond-

ing Ce values for each type of biomass, as shown in

Fig. 7. It can be seen that the shape of the two

adsorption isotherms is convex. Notice the different

scales on the y-axis of Figs. 7a and b. It is evident that

0

20

40

60

80

100

120

0 100 200 300 400 500

0

100

200

300

400

500

0 50 100 150 200 250 300

Fresh biomass

Immobilized biomass

Ce (mg/l)

Ce (mg/l)

q e (m

g/g)

q e (m

g/g)

(a)

(b)

Fig. 7. Equilibrium data of As(III) uptake by (a) fresh biomass

and (b) immobilized biomass fitted with the Langmuir model.

IGCFIX is superior to fresh biomass in binding As(III)

over the entire range of solution phase equilibrium

concentration tested. For example, at Ce ¼ 10mg/l, the

uptake capacity of IGCFIX is about five times that of

fresh biomass while at Ce ¼ 200mg/l the IGCFIX to

fresh biomass uptake capacity ratio climbs to about 7.

This means that a much smaller amount of IGCFIX is

needed compared to fresh biomass to remove a given

amount of As(III).

The experimental adsorption isotherms shown in Fig. 7

can be expressed in terms of mathematical models.

Because the shape of the two isotherms conforms to

the convex type, the simple Langmuir model with two

adjustable parameters is expected to provide a good fit

to the experimental data. The Langmuir model is

qe ¼ qmbCe

1þ bCe, (4)

where qm is the maximum or saturation uptake capacity

of the adsorbent (mg/g) and b (l/mg) is a binding

constant that reflects the slope of the isotherm in the low

solution phase equilibrium concentration range. Eq. (4)

predicts that the equilibrium uptake of an adsorbent

increases with increasing solution phase equilibrium

concentration, finally leveling off at qm as the Ce term in

the denominator becomes dominant. Eq. (4) was fitted

to the isotherm data in Fig. 7 by a nonlinear regression

analysis to obtain the parameters qm and b. The best-fit

values of the parameters together with the R2 values are

listed in Table 2. The isotherm simulations from the

Langmuir equation are shown as lines in Fig. 7. Both the

simulations and R2 values suggest that very good

agreement was obtained with the experimental isotherm

data.

In addition to comparing biosorbent performance,

adsorption isotherms are useful for designing and

analyzing adsorption processes. The design/performance

equation of a batch adsorption system obeying the

Langmuir model can be derived by combining Eqs. (1),

(2), and (4). The resulting equation is given below:

PR ¼ 100 1�� 1

b� Ci þ qmw

� �þ

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi1b� C i þ qmw

� �2þ 4

Cib

q2Ci

0@

1A.

(5)

In this study the criterion for measuring the predictive

capability of Eq. (5) is the absolute relative error (ARE)

Table 2

Estimation of qm and b by nonlinear regression

Qm (mg/l) b (l/mg) R2

Fresh biomass 128.10 0.011 0.971

Immobilized biomass 704.11 0.008 0.965

ARTICLE IN PRESS

Table 3

Absolute relative errors (AREs) in Eq. (5)’s estimates of the

percent removal data of Fig. 5

Experimental conditions ARE (%)

Ci (mg/l) w (g/l)

Fresh biomass 100 5 4.7

100 10 0.1

100 20 1.4

100 40 1.8

Immobilized biomass 100 5 3.5

100 10 1.8

100 20 0.9

100 40 0.4

70

80

90

100

10 25 50 100

As(

III)

perc

ent r

emov

al

Ca Mg Fe(III)

Initial concentration (mg/l)

Fig. 8. Effect of Ca, Mg, and Fe(III) on As(III) removal by

immobilized biomass.

C.T. Kamala et al. / Water Research 39 (2005) 2815–28262822

as defined in the following equation:

ARE ¼ 100PRexp � PRcal

PRexp

��������, (6)

where PRexp and PRcal denote experimental and

calculated percent removal. Table 3 shows AREs in

Eq. (5)’s estimates of the percent removal data of Fig. 5.

The calculations are based on the qm and b estimates

listed in Table 2. Note that the equilibrium data of Fig. 5

were not used to estimate qm and b in the nonlinear

curve-fitting exercise. For both types of biomass, the

predicted values of percent removal are very close to the

experimental values. The small AREs (maximum

AREo5%) are indicative of the good quality of these

predictions.

3.2.6. Effect of background ions

Ionic species such as Ca, Mg, and Fe are usually

present in drinking water and groundwater contami-

nated with arsenic (Smedley and Kinniburgh, 2002).

These ions may interfere in the uptake of arsenic by

adsorbent through competitive binding or complexa-

tion. Batch equilibrium experiments were conducted to

evaluate the influence of Ca, Mg, and Fe(III) on As(III)

removal by IGCFIX. Fig. 8 displays As(III) removal

obtained from experiments with Ci ¼ 10mg/l and

w ¼ 5 g/l at several initial concentrations of Ca, Mg,

and Fe(III). The bars in Fig. 8 denote the extent of

As(III) removal as a percentage of the original removal

when no background ions were present in solutions

containing As(III) (original percent removal ¼ �100%).

Within the initial concentration range of 10–100mg/l,

Ca and Mg had no noticeable effect on As(III) removal

by IGCFIX. On the other hand, when both As(III) and

Fe(III) were present in solution, As(III) removal

decreased with increasing Fe(III) concentration. The

presence of Fe(III) at an initial concentration of 10mg/l

reduced the original percent removal slightly, by about

3%. Increasing the initial Fe(III) concentration to

100mg/l caused the percent removal of As(III) to drop

to about 85%. From a practical standpoint, the

detrimental effect of Fe(III) on As(III) removal would

not pose much of a problem because the iron contents of

natural waters are usually much lower than 100mg/l.

As has been discussed earlier, based on the evidence

from the FT-IR analysis it is postulated that As(III)

removal is due to chelation/complexation by the

carboxyl groups of G. cambogia biomass. A previous

study has shown that carboxyl groups present in a

seaweed biosorbent participated in the uptake of Fe(III)

ions by chelation (Figueira et al., 1999). Furthermore,

citrate readily forms complexes with Fe(III) (Hug et al.,

1997). As pointed out earlier, hydroxycitric acid, which

has a chemical structure similar to that of citric acid, is

the major organic acid found in G. cambogia. Reduction

of As(III) removal in the presence of Fe(III) may thus be

attributed to competition between As(III) and Fe(III)

for the same carboxyl sites on hydroxycitric acid. Since

Ca and Mg are able to bind to carboxyl sites through ion

exchange, they can in principle compete with As(III) for

the same carboxyl sites on G. cambogia biomass.

However, the results in Fig. 8 suggest that Ca and Mg

compete weakly with As(III) which interacts with

carboxyl sites through chelation/complexation.

3.2.7. Removal of arsenic in groundwater samples

Batch equilibrium experiments were conducted to

assess the ability of fresh biomass and IGCFIX to

remove As(III) in groundwater samples collected from

three arsenic-contaminated sites in India. A biosorbent

dosage of 5 g/l was used in all batch tests. A series of

groundwater samples collected from the West Bengal

area (Das et al., 1996), the industrial region of

Patancheru near Hyderabad (Chandra Sekhar et al.,

2003a), and the state of Chhathisgarh (Pandey et al.,

2002) were analyzed for their As(III), As(V), and iron

contents. The results are tabulated in Table 4. It can be

seen that As(III) concentration ranges from a minimum

ARTICLE IN PRESSC.T. Kamala et al. / Water Research 39 (2005) 2815–2826 2823

of 1.1mg/l to a maximum of 7.9mg/l. Table 5 compares

measured and predicted values of As(III) percent

removal for the two types of biomass. The predicted

values were calculated using the qm and b estimates listed

in Table 2. Under the experimental conditions em-

ployed, almost quantitative removal of As(III) by

IGCFIX was observed in all cases. The predicted

percent removal values are in good agreement with the

measured values. In the case of fresh biomass, the extent

of As(III) removal is about 75% for the five ground-

water samples collected from the West Bengal and

Patancheru areas. The fresh biomass performed better in

removing As(III) in the four groundwater samples

collected from the Chhathisgarh region, yielding

87–90% removal. Interestingly, the predicted values of

percent removal are very close to the measured values

for the Chhathisgarh samples ðARE ¼ 0:422:6%Þ but

are noticeably higher than the measured values for the

Table 4

Arsenic and iron contents of groundwater samples collected

from three regions in India known to suffer from arsenic

contamination

Sampling site Sample

number

As(III)

(mg/l)

As(V)

(mg/l)

Fe

(mg/l)

West bengal W1 7.9 17.4 3.8

W2 6.2 19.8 6.2

Patancheru P1 3.3 4.0 0.3

P2 1.1 2.1 1.8

P3 1.9 2.1 4.1

Chhathisgarh C1 1.9 7.2 2.6

C2 1.1 4.2 2.9

C3 3.4 11.1 3.2

C4 2.0 6.0 4.1

Table 5

Measured and predicted values of As(III) percent removal for ground

Sample number Ci (mg/l) Fresh biomass

Percent removal

Measured Predicted

W1 7.9 73.6 87.5

W2 6.2 75.1 87.5

P1 3.3 73.9 87.5

P2 1.1 74.3 87.6

P3 1.9 75.7 87.5

C1 1.9 89.9 87.5

C2 1.1 88.5 87.6

C3 3.4 87.2 87.5

C4 2.0 89.0 87.5

samples collected from the other two areas

ðARE ¼ 15:6218:8%Þ.

The excellent agreement between the measured and

predicted values of As(III) removal suggests that As(III)

removal in groundwater of Chhathisgarh under field

conditions can be predicted with confidence using the

best-fit qm and b values for the two types of biomass

estimated under laboratory conditions. The deviations

between model predictions and measured values of

percent removal for the groundwater samples of West

Bengal and Patancheru are open to two interpretations.

First, the presence of As(V) species or iron in the

samples may have suppressed As(III) removal, and

second, the lower than expected As(III) removal may be

due to interfering effects of some unidentified chemical

species. The former is not likely to be the main reason

because similar levels of As(V) were also present in the

Chhathisgarh samples and the concentration range of

iron, as shown in Table 4 (0.3–6.2mg/l), was too low to

exert any significant effect on As(III) removal. It is

therefore plausible that the lower than expected As(III)

removal by fresh biomass may be due to the presence of

some chemical species that can either compete with

As(III) for carboxyl sites or impede As(III) uptake. Such

negative effects are much less pronounced or nondetect-

able in the case of IGCFIX owing to its significantly

larger As(III) uptake capacity compared with fresh

biomass. Work is currently underway to identify the

various chemical species present in the groundwater

samples examined in this study and to assess the ability

of fresh biomass and IGCFIX to remove As(V) species.

These results will be reported in future communications.

3.2.8. Elution tests

One of the objectives of biomass immobilization is to

enhance the reusability of the resulting immobilized

water samples treated with fresh biomass and IGCFIX

Immobilized biomass

ARE (%) Percent removal ARE (%)

Measured Predicted

18.8 �100 96.6 3.4

16.5 �100 96.6 3.4

18.4 �100 96.6 3.4

17.8 �100 96.6 3.4

15.6 �100 96.6 3.4

2.6 �100 96.6 3.4

1.1 �100 96.6 3.4

0.4 �100 96.6 3.4

1.6 �100 96.6 3.4

ARTICLE IN PRESS

40

60

80

100

0.025 0.05 0.1 0.2

As(

III)

perc

ent e

lute

d

HCl

HNO3

NaCl

Na2CO3

NaOH

Concentration (M)

0.15

Fig. 9. Elution of bound As(III) from immobilized biomass

using various eluting agents.

C.T. Kamala et al. / Water Research 39 (2005) 2815–28262824

biomass. Reusing immobilized biomass requires that the

bound species be eluted from the biomass. Preliminary

elution tests were conducted using the batch technique

to select an eluting agent that could desorb all the bound

As(III) from IGCFIX. Reusability was assessed using a

packed column set-up described below. IGCFIX beads

were first loaded with As(III) in batch uptake experi-

ments. Batch elution experiments were then carried out

to desorb the bound As(III) from IGCFIX using five

different eluting agents, namely, HCl, HNO3, NaCl,

Na2CO3, and NaOH at a range of concentrations. The

batch elution results are shown in Fig. 9. It is evident

that the two mineral acids, HCl and HNO3, performed

poorly over the range of concentration tested. On the

other hand, close to complete elution of the bound

As(III) could be achieved using 0.2M NaCl, 0.1M

Na2CO3 or 0.05M NaOH. Since high concentrations of

eluting agent may have an adverse effect on reusability,

it was decided to employ 0.05M NaOH for further

testing using a column packed with IGCFIX under

continuous-flow conditions.

4. Column studies

Full-scale adsorption processes are often operated

using continuous-flow fixed-bed columns. Adsorbents

possessing strong mechanical strength and durability are

required for column application in order to avoid

operational problems such as clogging and pressure

drop fluctuations. For this reason, fresh G. cambogia

biomass has been immobilized in a solid matrix to

enhance its mechanical strength. To be viable for

practical application, immobilized biomass should retain

its uptake capacity over multiple cycles of use. In this

study five cycles of As(III) loading and elution were

conducted using a laboratory-scale column packed with

IGCFIX beads. Each cycle consisted of loading with an

aqueous As(III) solution (Ci ¼ 100mg/l) and elution of

the bound As(III) with 0.05M NaOH. In each cycle,

loading was terminated before As(III) breakthrough

occurred at the column exit. The amount of As(III)

loaded onto the column was estimated from the known

amount of As(III) solution applied. Similarly, the

amount of As(III) eluted was estimated from the known

amount of NaOH solution used in the elution step.

Comparison of the amount of As(III) loaded and eluted

within each cycle indicates that As(III) could be

recovered quantitatively in each cycle under continu-

ous-flow conditions. These preliminary experiments

demonstrate that IGCFIX was compatible with column

operation.

5. Conclusion

In this study we have demonstrated the ability of G.

cambogia to remove As(III) in synthetic solutions as well

as groundwater samples collected from arsenic-contami-

nated sites in India. The following specific onclusions

were reached:

�

FT-IR results indicated that carboxyl groups were

the main sites responsible for As(III) binding on G.

cambogia biomass. The carboxyl groups would be

mainly those present in hydroxycitric acid which is

the principal organic acid found in G. cambogia.

�

The immobilized form of biomass showed excellent

As(III) sequestering capabilities compared with fresh

biomass. It was demonstrated that the immobiliza-

tion method enhanced the kinetic property as well as

the equilibrium uptake capacity of fresh biomass.

�

The extent of As(III) removal by fresh and immobi-

lized biomass was insensitive to the solution pH. The

pH range of 6–8 was found to provide optimum

As(III) removal. The presence of background ions

such as Ca and Mg at concentrations up to 100mg/l

and Fe(III) at 10mg/l had no significant effect on

As(III) removal.

�

The effects of adsorbent dosage and initial As(III)

concentration on As(III) uptake by fresh and

immobilized biomass were characterized using the

batch procedure and the resulting equilibrium data

quantified and validated in terms of the Langmuir

isotherm model. Adsorption isotherms derived under

these laboratory conditions were shown to be a useful

tool for predicting the extent of As(III) removal in

groundwater samples collected from a site contami-

nated with arsenic.

�

Batch elution tests revealed that close to complete

elution of bound As(III) from immobilized biomass

could be achieved using aqueous solutions of NaCl,

Na2CO3, or NaOH. Of the three eluting agents,

NaOH appeared to be the most desirable, requiring

ARTICLE IN PRESSC.T. Kamala et al. / Water Research 39 (2005) 2815–2826 2825

the lowest solution strength to achieve complete

elution.

�

Fixed-bed column studies demonstrated the robust-

ness of immobilized biomass in five cycles of As(III)

loading and elution. It was established that 0.05M

NaOH could be used to elute bound As(III) without

causing any damage to the uptake capacity of

immobilized biomass.

Acknowledgements

Dr. K.V. Raghavan, Director, Indian Institute of

Chemical Technology (IICT), and Dr. V. Balaram,

Head, Geochemistry Department, National Geophysical

Research Institute (NGRI), encouraged and supported

this work. Fellowships provided by the Council of

Scientific and Industrial Research to C.T. Kamala and

N.S. Chary are gratefully acknowledged.

References

Bissen, M., Frimmel, F.H., 2003. Arsenic—a review. Part II:

Oxidation of arsenic and its removal in water treatment.

Acta Hydrochim. Hydrobiol. 31, 97–107.

Chakravarty, S., Dureja, V., Bhattacharyya, G., Maity, S.,

Bhattacharjee, S., 2002. Removal of arsenic from ground-

water using low cost ferruginous manganese ore. Water Res

36, 625–632.

Chandra Sekhar, K., Chary, N.S., Kamala, C.T., Venkatesh-

warar Rao, J., Balaram, V., Anjaneyulu, Y., 2003a. Risk

assessment and pathway study of arsenic in industrially

contaminated sites of Hyderabad: a case study. Environ.

Int. 29, 601–611.

Chandra Sekhar, K., Kamala, C.T., Chary, N.S., Anjaneyulu,

Y., 2003b. Removal of heavy metals using a plant biomass

with reference to environmental control. Int. J. Miner.

Processing, 68, 37–45.

Chandra Sekhar, K., Kamala, C.T., Chary, N.S., Sastry,

A.R.K., Nageswara Rao, T., Vairamani, M., 2004. Re-

moval of lead from aqueous solutions using an immobilized

biomaterial derived from a plant biomass. J. Hazard. Mat.

B 108, 111–117.

Dambies, L., Vincent, T., Guibal, E., 2002. Treatment of

arsenic-containing solutions using chitosan derivatives:

uptake mechanism and sorption performances. Water Res.

36, 3699–3710.

Dambies, L., 2004. Existing and prospective sorption technol-

ogies for the removal of arsenic in water. Sep. Sci. Technol.

39, 603–627.

Das, D., Samanta, G., Mandal, B.K., Chowdhury, T.R.,

Chanda, C.R., Chowdhury, P.P., Basu, G.K., Chakraborti,

D., 1996. Arsenic in groundwater in six districts of West

Bengal, India. Environ. Geochem. Health 18, 5–15.

Daus, B., Wennrich, R., Weiss, H., 2004. Sorption materials for

arsenic removal from water: a comparative study. Water

Res. 38, 2948–2954.

DeMarco, M.J., SenGupta, A.K., Greenleaf, J.E., 2003.

Arsenic removal using a polymeric/inorganic hybrid sor-

bent. Water Res. 37, 164–176.

Dousova, B., Machovic, V., Kolousek, D., Kovanda, F.,

Dornicak, V., 2003. Sorption of As(V) species from aqueous

systems. Water Air Soil Pollut. 149, 251–267.

Elizalde-Gonzalez, M.P., Mattusch, J., Einicke, W.-D., Wenn-

rich, R., 2001. Sorption on natural solids for arsenic

removal. Chem. Eng. J. 81, 187–195.

Figueira, M.M., Volesky, V., Mathieu, H.J., 1999. Instrumental

analysis study of iron species biosorption by Sargassum

biomass. Environ. Sci. Technol. 33, 1840–1846.

Genc--Fuhrman, H., Tjell, J.C., McConchie, D., 2004a.

Increasing the arsenate adsorption capacity of neutralized

red mud (bauxsol). J. Colloid Interface Sci. 271, 313–320.

Genc--Fuhrman, H., Tjell, J.C., McConchie, D., 2004b.

Adsorption of arsenic from water using activated neutra-

lized red mud. Environ. Sci. Technol. 38, 2428–2434.

Ghimire, K.N., Inoue, K., Makino, K., Miyajima, T., 2002.

Adsorptive removal of arsenic using orange juice residue.

Sep. Sci. Technol. 37, 2785–2799.

Ghimire, K.N., Inoue, K., Yamaguchi, H., Makino, K.,

Miyajima, T., 2003. Adsorptive separation of arsenate and

arsenite anions from aqueous medium by using orange

waste. Water Res. 37, 4945–4953.

Hug, S.J., Laubscher, H.-U., James, B.R., 1997. Iron(III)

catalyzed photochemical reduction of chromium(VI) by

oxalate and citrate in aqueous solutions. Environ. Sci.

Technol. 31, 160–170.

Jeffers, T.H., Ferguson, C.R., Bennetm, P.G., 1991. Biosorp-

tion of metal contaminants using immobilized biomass—a

laboratory study. Report of Investigations 9340, United

States Bureau of Mines.

Jena, B.S., Jayaprakasha, G.K., Singh, R.P., Sakariah, K.K.,

2002. Chemistry and biochemistry of (–)-hydroxycitric acid

from Garcinia. J. Agric. Food Chem. 50, 10–22.

Katsoyiannis, I.A., Zouboulis, A.I., 2002. Removal of arsenic

from contaminated water sources by sorption onto iron-

oxide-coated polymeric materials. Water Res. 36,

5141–5155.

Kim, Y., Kim, C., Choi, I., Rengaraj, S., Yi, J., 2004. Arsenic

removal using mesoporous alumina prepared via a templat-

ing method. Environ. Sci. Technol. 38, 924–931.

Lewis, Y.S., Neelakantan, S., 1965. (–)-Hydroxycitric acid—the

principal acid in the fruits of Garcinia cambogia desr.

Phytochemistry 4, 619–625.

Loukidou, M.X., Matis, K.A., Zouboulis, A.I., Liakopoulou-

Kyriakidou, M., 2003. Removal of As(V) from wastewaters

by chemically modified fungal biomass. Water Res 37,

4544–4552.

Mcafee, B.J., Gould, W.D., Nadeau, J.C., da Costa, A.C.A.,

2001. Biosorption of metal ions using chitosan, chitin, and

biomass of Rhizopus oryzae. Sep. Sci. Technol. 36,

3207–3222.

Pandey, P.K., Yadav, S., Nair, S., Bhui, A., 2002. Arsenic

contamination of the environment: a new perspective from

central-east India. Environ. Int. 28, 235–245.

Pandey, P.K., Yadav, S., Nair, S., Pandey, M., 2004. Sampling

and preservation artifacts in arsenic analysis: implications

for public health issues in developing countries. Curr. Sci.

86, 1426–1432.

ARTICLE IN PRESSC.T. Kamala et al. / Water Research 39 (2005) 2815–28262826

Rau, I., Gonzalo, A., Valiente, M., 2003. Arsenic(V) adsorption

by immobilized iron mediation. Modeling of the adsorption

process and influence of interfering anions. React. Funct.

Polym. 54, 85–94.

Sadka, A., Artzi, B., Cohen, L., Dahan, E., Hasdai, D., Tagari,

E., Erner, Y., 2000. Arsenite reduces acid content in citrus

fruit, inhibits activity of citrate synthase but induces its gene

expression. J. Am. Soc. Hortic. Sci. 125, 288–293.

Say, R., Yilmaz, N., Denizli, A., 2003a. Biosorption

of cadmium, lead, mercury, and arsenic ions by the

fungus Penicillium purpurogenum. Sep. Sci. Technol. 38,

2039–2053.

Say, R., Yilmaz, N., Denizli, A., 2003b. Removal of heavy

metal ions using the fungus Penicillium canescens. Adsorp.

Sci. Technol. 21, 643–650.

Seki, H., Suzuki, A., Maruyama, H., 2005. Biosorption of

chromium(VI) and arsenic(V) onto methylated yeast bio-

mass. J. Colloid Interface Sci. 281, 261–266.

Singh, T.S., Pant, K.K., 2004. Equilibrium, kinetics and

thermodynamic studies for adsorption of As(III) on

activated alumina. Sep. Purif. Technol. 36, 139–147.

Smedley, P.L., Kinniburgh, D.G., 2002. A review of the source,

behaviour and distribution of arsenic in natural waters.

Appl. Geochem. 17, 517–568.

Teixeira, M.C., Ciminelli, V.S.T., 2005. Development of a

biosorbent for arsenite: structural modeling based on X-ray

spectroscopy. Environ. Sci. Technol. 39, 895–900.

Thirunavukkarasu, O.S., Viraraghavan, T., Subramanian, K.S.,

Chaalal, O., Islam, M.R., 2005. Arsenic removal in drinking

water—impacts and novel removal technologies. Energy

Sources 27, 209–219.

Vaishya, R.C., Gupta, S.K., 2004. Modeling arsenic(V) removal

from water by sulfate modified iron-oxide coated sand

(SMIOCS). Sep. Sci. Technol. 39, 645–666.

Wang, J.S., Wai, C.M., 2004. Arsenic in drinking water—a

global environmental problem. J. Chem. Educ. 81, 207–213.

Xu, Y., Nakajima, T., Ohki, A., 2002. Adsorption and removal

of arsenic(V) from drinking water by aluminum-loaded

Shirasu-zeolite. J. Hazard. Mat. B 92, 275–287.

Zeng, L., 2003. A method for preparing silica-containing

iron(III) oxide adsorbents for arsenic removal. Water Res.

37, 4351–4358.

Zhang, Y., Yang, M., Huang, X., 2003. Arsenic(V) removal

with a Ce(IV)-doped iron oxide adsorbent. Chemosphere

51, 945–952.

Zouboulis, A.I., Katsoyiannis, I.A., 2002. Arsenic removal

using iron oxide loaded alginate beads. Ind. Eng. Chem.

Res. 41, 6149–6155.