AAPPPPEENNDDIIXX--II

PPUUBBLLIICCAATTIIOONNSS

Published

1. Suresh Kumar, K., Ganesan, K., & Subba Rao, P. V. (2007). Antioxidant potential of

solvent extracts of Kappaphycus alvarezii ( Doty) Doty- an edible seaweed. Food

Chemistry (doi. 10.1016/j.foodchem.2007.08.016).

2. Suresh Kumar, K., Ganesan, K., & Subba Rao, P. V. (2007). Phycoremediation of

heavy metals by three-color form of Kappaphycus alvarezii. Journal of Hazardous

Materials. 143, 1-2, 590-592

3. Suresh Kumar, K., Ganesan, K., & Subba Rao, P. V. (2007). Heavy metal chelation by

non-living biomass of three color forms of Kappaphycus alvarezii (Doty) Doty.

Journal of Applied Phycology (doi.10.1007/s10811-007-9181-8).

4. Subba Rao, P. V., Vaibhav A. Mantri., Ganesan, K., & Suresh Kumar, K. (2007).

Seaweeds as a human diet an emerging trend in the new Millennium. In: Advances in

Applied Phycology, R. K. Gupta and V. D. Pandey (Eds.), (Daya Publishing House,

New Delhi), pp. 85-96.

5. Vaibhav A. Mantri., Subba Rao, P.V., Thakur, M.C., Ganesan, K., & K. Suresh Kumar

(2005). Some observations on Trichosolen mucronatus (Børgesen) Taylor

[Bryopsidaceae], from Gujarat: A species of rare occurrence. National Academy

Science letters 28, (9& 10): 341 – 344.

Communicated

1. Suresh Kumar, K., Ganesan, K., & Subba Rao, P. V. (2007). Functional properties, FT-

IR and FT-Raman spectroscopy of protein concentrate of Kappaphycus alvarezii

(Doty) Doty-an edible seaweed (Journal of Agricultural Food Chemistry).

2. Suresh Kumar, K., Ganesan, K., & Subba Rao, P. V. (2007). Chemical and mineral

composition of three-color forms of Kappaphycus alvarezii (Botanica Marina).

3. Ganesan, K., Suresh Kumar, K., & Subba Rao, P. V. (2007). Free radical-scavenging

activity of aqueous extract of Padina tetrastromatica (Hauck) ( Fitoterapia).

4. Subba Rao, P.V., Suresh Kumar, K., Ganesan, K., & and Thakur, M.C. (2007).

Feasibility of cultivation of Kappaphycus alvarezii Doty (Doty) at different localities

on Northwest coast of India (Aquaculture Research).

5. Ganesan, K., Suresh Kumar, K., & Subba Rao, P. V. (2007). Antioxidant potential of

various solvent extracts of edible seaweed Enteromorpha compressa. (Journal of

Applied Phycology).

Available online at www.sciencedirect.com

www.elsevier.com/locate/foodchem

Food Chemistry 107 (2008) 289–295

FoodChemistry

Antioxidant potential of solvent extracts of Kappaphycusalvarezii (Doty) Doty – An edible seaweed

K. Suresh Kumar, K. Ganesan, P.V. Subba Rao *

Marine Biotechnology and Ecology Discipline, Central Salt and Marine Chemicals Research Institute (CSIR), Bhavnagar – 364 002, Gujarat, India

Received 11 May 2007; received in revised form 30 June 2007; accepted 6 August 2007

Abstract

Various solvent extracts of Kappaphycus alvarezii, an edible red seaweed (family Solieriaceae) were screened for total phenol contentand antioxidant activity using 1,1-diphenyl-2-picrylhydrazyl (DPPH), ferrous ion chelating activity, reducing power and antioxidantactivity assays in a linoleic acid system with ferrothiocyanate reagent (FTC). The total phenol content of different extracts of K. alvarezii

varied from 0.683 ± 0.040% to 2.05 ± 0.038%. The radical-scavenging activity of ethanol extract was, as IC50 3.03 mg ml�1, whereas thatof the water extract was IC50 4.76 mg ml�1. Good chelating activity was recorded for methanol extract (IC50 3.08 mg ml�1) wherein67.0 ± 0.924% chelation was obtained using 5.0 mg ml�1 of extract. The reducing power of the samples was in the following order:BHT > methanol > ethanol > ethyl acetate > water > hexane. But, in the linoleic acid system, the ethanol extract proved superior tothe synthetic antioxidants butylated hydroxytoluene (BHT). Hence, these extracts could be considered as natural antioxidants andmay be useful for curing diseases arising from oxidative deterioration.� 2007 Elsevier Ltd. All rights reserved.

Keywords: Kappaphycus alvarezii; Antioxidant activity; Total phenolics; Free radicals; Reducing power

1. Introduction

All living organisms contain complex systems of antiox-idant enzymes. Some of these systems, e.g. the thioredoxinsystem, are conserved throughout evolution and arerequired for life. Antioxidants in biological systems havemultiple functions, including defending against oxidativedamage and participating in the major signalling pathwaysof cells. One major action of antioxidants in cells is to pre-vent damage caused by the action of reactive oxygen spe-cies. Reactive oxygen species include hydrogen peroxide(H2O2), the superoxide anion (O�2 ), and free radicals, suchas the hydroxyl radical (�OH). These molecules are unstableand highly reactive, and can damage cells by chain reac-tions, such as lipid peroxidation, or formation of DNA

0308-8146/$ - see front matter � 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.foodchem.2007.08.016

* Corresponding author.E-mail address: pvsubbarao@csmcri.org (P.V.S. Rao).

adducts that could cause cancer-promoting mutations orcell death. In order to reduce or prevent this damage, allcells invariably contain antioxidants.

Lipid oxidation by reactive oxygen species (ROS), suchas superoxide anion, hydroxyl radicals, and hydrogen per-oxide, causes a decrease in nutritional value of lipids, intheir safety and appearance. In addition, it is the predom-inant cause of qualitative decay of foods, which leads torancidity, toxicity, and destruction of biochemical compo-nents important in physiologic metabolism. Free radical-mediated modification of DNA, proteins, lipids and smallcellular molecules are associated with a number of patho-logical processes, including atherosclerosis, arthritis, diabe-tes, cataractogenesis, muscular dystrophy, pulmonarydysfunction, inflammatory disorders, ischemiareperfusiontissue damage, and neurological disorders, such as Alzhei-mer’s disease (Frlich & Riederer, 1995).

Antioxidants are classified by the products they formon oxidation (these can be antioxidants themselves, inert,

290 K.S. Kumar et al. / Food Chemistry 107 (2008) 289–295

or pro-oxidant), by what happens to the oxidation prod-ucts (the antioxidant may be regenerated by differentantioxidants or, in the case of ‘‘sacrificial” antioxidants,its oxidized form may be broken down by the organism)and how effective the antioxidant is against specific freeradicals. Several synthetic antioxidants, such as butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT),and tert-butylhydroquinone (TBHQ), are commerciallyavailable and currently used. However, these antioxidantshave been restricted for use in foods as they are sus-pected to be carcinogenic. Some toxicological studieshave also implicated the use of these synthetic antioxi-dants in promoting the development of cancerous cellsin rats. These findings, together with consumers’ interestsin natural food additives, have reinforced the efforts forthe development of alternative antioxidants of naturalorigin (Huang & Wang, 2004). An immense number ofmarine flora and fauna are reported to have a wide spec-trum of interesting biological properties. In folk medi-cine, seaweeds have been used for a variety of remedialpurposes, e.g. for the treatment of eczema, gallstone,gout, crofula, cooling agent for fever, menstrual trouble,renal problems and scabies (Chapman & Chapman,1976).

Seaweeds are rich in polysaccharides, minerals, proteinsand vitamins. Documented antioxidant activity would ele-vate their value in the human diet as food and pharmaceu-tical supplements (Yan, Nagata, & Fan, 1998). Few reportsare available on the antioxidant potential of seaweeds(Jimenez-Escrig, Jimenez-Jimenez, Pulido, & Saura-Cali-xto, 2001). Ismail and Hong (2002) reported antioxidantactivity of four commercial edible seaweeds, namely Nori(Porphyra sp.), Kumbu (Laminaria sp.), Wakame (Undaria

sp.) and Hijiki (Hijikia sp.).The Rhodophyta (red algae) are a distinct eukaryotic

lineage, characterized by the accessory photosynthetic pig-ments phycoerythrin, phycocyanin and allophycocyaninsarranged in phycobilisomes. They contain a large assem-blage of species that predominate in the coastal and con-tinental shelf areas of tropical, temperate and cold-waterregions. Red algae are ecologically significant as primaryproducers, providers of structural habitat for other mar-ine organisms, and they play an important role in the pri-mary establishment and maintenance of coral reefs. Somered algae are economically important as providers of foodand gels (Wilson, 2000). For this reason, extensive farm-ing and natural harvest of red algae occur in numerousareas of the world. Kappaphycus alvarezii, an economi-cally important red tropical seaweed, which is highlydemanded for its cell wall polysaccharide, is the mostimportant source of kappa carrageenan. The worldproduction of Kappaphycus species is approximately28000 tons per annum. This seaweed accounts for thelargest consumption worldwide (McHugh, 1987). It is eas-ily accessible, in huge amounts, for food and pharmaceu-tical applications. The present study deals withantioxidant properties of K. alvarezii.

2. Materials and methods

2.1. Collection of samples

K. alvarezii was collected from a cultivation site at PortOkha (L 22�28.5280N; L 069�04.3220E) located on the northwest coast of India during April, 2006. The sample wasthoroughly washed with seawater to remove epiphytesand dirt particles, followed by shade-drying for two days.It was then brought to the laboratory, oven-dried at 70 �Cfor 4 h to obtain a constant weight and pulverized in thegrinder (size 2 mm). This sample was used for determina-tion of phenolic content, as well as for antioxidant studies.The chemicals used in these studies were of analytical grade.

2.2. Preparation of extracts

The pulverized moisture-free sample (20 g) wasextracted with 200 ml of individual solvents using a Soxhletextractor. The extraction was repeated many times toobtain a sizable quantity of extract. Consequently, theextract was concentrated in a rotary evaporator at 40 �C.Different solvents were used for the preparation of extractsto determine the antioxidant efficacy of K. alvarezii. All theexperiments were conducted in triplicate.

2.3. Determination of total phenol

Total phenolic content was estimated by Folin–Ciocal-teau method (Singleton & Rossi, 1965). To 6.0 ml ofdouble-distilled water, 0.1 ml of sample and 0.5 ml ofFolin–Ciocalteau reagent were mixed, followed by the addi-tion of 1.5 ml of Na2CO3 (20 g 100 ml�1 water) and thevolume was made up to 10.0 ml with distilled water. Afterincubation for 30 min at 25 �C, the absorbance wasmeasured at 760 nm and the total phenolic content was cal-culated with a gallic acid standard and expressed as a per-centage of total phenols obtained on a dry weight basis.

2.4. DPPH radical scavenging assay

DPPH�-scavenging potential of different fractions wasmeasured, based on the scavenging ability of stable 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals by K. alvarezii

antioxidants. The ability of extracts to scavenge DPPH rad-icals was determined by the method of Blois (1958). Briefly,1 ml of 1 mM methanolic solution of DPPH� was mixedwith 1 ml of extract solution (containing 0.5–5.0 mg ml�1

of dried extract). The mixture was then vortexed vigorouslyand left for 30 min at room temperature in the dark. Theabsorbance was measured at 517 nm and activity wasexpressed as percentage DPPH�-scavenging activity relativeto the control, using the following equation:

% Radical scavenging activity

¼ AControl � ASample=AControl

� �� 100

K.S. Kumar et al. / Food Chemistry 107 (2008) 289–295 291

2.5. Ferrous ion-chelating activity

Iron-chelating abilities of methanol, ethanol and ethylacetate extracts of K. alvarezii were used for the presentinvestigation. The chelating of ferrous ions by the extractsand standards was estimated by the method of Dinis,Madeira and Almeida (1994). Extracts were added to asolution of 2 mM FeCl2 (0.05 ml). The reaction was initi-ated by the addition of 5 mM ferrozine (0.2 ml) and themixture was shaken vigorously and left standing at roomtemperature for 10 min. After the mixture had reachedequilibrium, the absorbance of the solution was then mea-sured at 562 nm. The percentage inhibition of ferrozine–Fe2+ complex formation was determined using the follow-ing formula:

% Inhibition ¼ 1� A1Sample=A0Control

� �� 100

where A0 was the absorbance of the control and A1 was theabsorbance in the presence of the sample extracts and stan-dards. The control contained FeCl2 and ferrozine, withcomplex formation molecules.

2.6. Reducing power

Extracts of K. alvarezii were prepared using methanol,ethanol, water, ethyl acetate and hexane. The reductivepotential of extracts was determined by the method ofOyaizu (1986). The different concentrations of extracts(0.5–25 mg ml�1) were mixed with phosphate buffer(2.5 ml, 0.2 M, pH 6.6) and potassium ferricyanide[K3Fe(CN)6] (2.5 ml, 1%). The mixture was incubated at50 �C for 20 min. A portion (2.5 ml) of trichloroacetic acid(10%) was added to the mixture, which was then subjectedto centrifugation (10 min, 1000g). The upper layer of solu-tion (2.5 ml) was mixed with distilled water (2.5 ml) andFeCl3 (0.5 ml, 0.1%), and the absorbance was measuredat 700 nm. Higher absorbance of the reaction mixture indi-cated greater reductive potential.

2.7. Antioxidant activity in the linoleic acid system with

ferrothiocyanate reagent (FTC)

Ethanolic extract of K. alvarezii was subjected to theassay adopted by Osawa and Namaki (1983). The extract(4 mg) was dissolved in 99.5% ethanol and mixed with2.5% linoleic acid in 99.5% ethanol (4.1 ml), 0.05 M phos-phate buffer (pH 7, 8 ml) and distilled water (3.9 ml) andkept in screw-cap containers under dark conditions at40 �C; 0.1 ml of this solution was added to 9.7 ml of 75%ethanol and 0.1 ml of 30% ammonium thiocyanate. After3 min, 0.1 ml of 0.02 M ferrous chloride in 3.5% hydrochlo-ric acid was added to the reaction mixture, the absorbanceof the red colour was measured at 500 nm in the spectro-photometer every two days. The control and standard weresubjected to the same procedure except that for the control,there was no addition of sample and, for the standard,4 mg of sample was replaced with 4 mg of butylated

hydroxy toluene (BHT), used as a positive control. Absor-bance was measured at intervals of 2 days. The percentinhibition of linoleic acid peroxidation was calculated as:

Inhibition ð%Þ¼ 100� ðabsorbance increase of the sample½=absorbance increase of the controlÞ � 100�

The IC50 value represented the concentration of the com-pounds that caused 50% inhibition. All experiments werecarried out in triplicate.

2.8. Statistical analysis

For the extract, three samples were prepared for eachexperiment. The data were presented as mean ± standarddeviation.

3. Results and discussion

3.1. Antioxidant activity

The antioxidant activity is system-dependent. Moreover,it depends on the method adopted and the lipid systemused as substrate (Singh, Maurya, de Lampasona, & Cata-lan, 2006). Hence, the following different methods havebeen adopted in order to assess the antioxidative potentialof K. alvarezii extracts.

3.2. Total phenol content

A number of studies have focussed on the biologicalactivities of phenolic compounds, which are potential anti-oxidants and free radical-scavengers (Kahkonen et al.,1999; Rice-Evans, Miller, Bolwell, Bramley, & Pridham,1995; Sugihara, Arakawa, Ohnishi, & Furuno, 1999). Thetotal phenol content was maximum when a mixture ofchloroform and methanol (2:1) was used (2.05 ± 0.038%)followed by ethanol (1.94 ± 0.029%), methanol(1.79 ± 0.77%), n-propanol (1.40 ± 0.040%) and ethyl ace-tate (1.09 ± 0.597%). Extracts obtained using other sol-vents, namely acetone, n-hexane and chloroform, showed<1% total phenol content (Table 1).

3.3. Scavenging effect on 1,1-diphenyl-2-picrylhydrazyl

radical (DPPH�)

The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical is astable radical with a maximum absorbance at 517 nm thatcan readily undergo reduction by an antioxidant. Becauseof the ease and convenience of this reaction, it has nowwidespread use in the free radical-scavenging activityassessment (Brand-Williams, Cuvelier, & Benset, 1995).The radical-scavenging activity of K. alvarezii extract isshown in Fig. 1 and expressed as percentage reduction ofthe initial DPPH� absorption by the tested compound.The best radical-scavenging activity could be obtained in

Table 1Percent phenol content of K. alvarezii in various solvents

Solvents Total phenol (%)

Acetone 0.963 ± 0.058n-Propanol 1.40 ± 0.040Ethyl acetate 1.09 ± 0.597n-Hexane 0.83 ± 0.048Chloroform 0.683 ± 0.040Methanol 1.79 ± 0.77Ethanol 1.94 ± 0.029Chloroform:methanol (2:1) 2.05 ± 0.038

Values are means of three replicate determinations; SD, standarddeviation.

292 K.S. Kumar et al. / Food Chemistry 107 (2008) 289–295

the ethanol extract (IC50 3.03 mg ml�1), followed by meth-anol (IC50 4.28 mg ml�1). Extracts obtained using wateralso showed equivalent scavenging activity (IC50

4.76 mg ml�1). These values were lower than thoseobtained using BHT (IC50 2.83 mg ml�1), but the IC50 val-ues of the methanol and water extracts were comparablewith a-tocopherol (IC50 4.55 mg ml�1). The extracts of K.

alvarezii showed better radical-scavenging activity thandid the extract of Palmaria palmata (dulse) IC50 –12.5 mg ml�1 (Yuan, Carrington, & Walsh, 2005a), andpurified extract of Ecklonia cava IC50 – 5.49 � 103 lg ml�1

(c.f. Suja, Jayalekshmy, & Arumughan, 2005). Ragan andGlombitza (1986) reported the radical-scavenging activityof seaweeds to be mostly related to their phenolic contents.On the other hand, Siriwardhana, Lee, Kim, Ha, and Jeon(2003) and Lu and Foo (2000) reported a high correlationbetween DPPH radical-scavenging activities and total poly-phenolics r = (0.971). In the present study, the linearregression analysis of DPPH�-scavenging (i.e EC50 values)with the total phenol content (gallic acid equivalents) gavean r value of 0.937, showing statistically significant correla-tion. K. alvarezii is the main industrial source of carra-geenan (having alternating D-galactose 4-sulphate and3,6-anhydro D-galactose residues), which may also contrib-

Fig. 1. Antioxidant activities of different solvent extracts of K.

ute to the antioxidant potential of this seaweed. Compo-nents, such as low molecular weight polysaccharides,pigments, proteins or peptides, also influence the antioxi-dant activity (Siriwardhana et al., 2003).

3.4. Metal ion-chelating activity

All the extracts demonstrated reasonable ferrous ion che-lating efficacy (Fig. 2). The ascorbic acid extract demon-strated best ferrous chelating efficacy (IC50 2.88 mg ml�1)followed by methanol, ethanol and ethyl acetate (IC50

3.08, 3.83 and 4.38 mg ml�1, respectively). Iron is knownto generate free radicals through the Fenton & Haber-Weissreaction. Metal ion-chelating activity of an antioxidantmolecule prevents oxyradical generation and the conse-quent oxidative damage. Metal ion-chelating capacity playsa significant role in the antioxidant mechanism since itreduces the concentration of the catalyzing transition metalin LPO. It is reported that chelating agents that form r-bonds with a metal, are effective as secondary antioxidantssince they reduce the redox potential, thereby stabilizing theoxidized form of the metal ion (Srivastava, Harish, & Shiva-nandappa, 2006). Metal-binding capacities of dietary fibresare well known, e.g. the inhibitory effects on ferrous absorp-tion of algal dietary fibres such as carrageenan, agar andalginate, were reported (Harmuth-Hoene & Schelenz,1980). In this present study, the carrageenan might havecaused the decrease of ferrous ion in the assay system.

3.5. Measurement of reducing potential

The reducing power of K. alvarezii extracts was concen-tration-dependent (Fig. 3). As the concentration increasedfrom 0.5 to 5.0 mg ml�1, there was an increase in absor-bance with all the solvents except hexane. However, thereducing powers of the samples were found to be in the fol-lowing order: BHT (0.23–0.879) > methanol (0.07–

alvarezii determined as DPPH radical-scavenging activity.

Fig. 2. Ferrous ion-chelating activities of different solvent extracts of K. alvarezii.

Fig. 3. Reducing powers of K. alvarezii extracts, along with a synthetic antioxidant.

K.S. Kumar et al. / Food Chemistry 107 (2008) 289–295 293

0.74) > ethanol (0.333–0.44) > ethyl acetate (0.013–0.467) > water (0.017–0.193) > hexane (0.017–0.16). It isbelieved that antioxidant activity and reducing power arerelated. Reductones inhibit LPO by donating a hydrogenatom and thereby terminating the free radical chain reac-tion (Srivastava et al., 2006).

3.6. Antioxidant activity in a linoleic acid system with

ferrothiocyanate reagent (FTC)

Peroxyl radicals are formed by a direct reaction of oxy-gen with alkyl radicals. Decomposition of alkyl peroxidesalso results in peroxyl radicals. Peroxyl radicals are goodoxidizing agents, having more than 1000 mV of standardreduction potential (Decker, 1998). They can abstracthydrogen from other molecules with lower standard reduc-tion potentials. This reaction is frequently observed in the

propagation stage of lipid peroxidation. Cell membranesare phospholipid bilayers with extrinsic proteins and arethe direct target of lipid oxidation (Girotti, 1998). As lipidoxidation of cell membranes increases, the polarity of lipidphase surface charge and formation of protein oligomersincrease; and molecular mobility of lipids, number of SHgroups, and resistance to thermal denaturation decrease.Malonaldehyde, one of the lipid oxidation products, canreact with the free amino group of proteins, phospholipid,and nucleic acids, leading to structural modifications,which induce dysfunction of immune systems. The antiox-idant effects of K. alvarezii extract and BHT on the perox-idation of linoleic acid were investigated and the results arepresented in Fig. 4. The absorbance ranges recorded forcontrol, BHT and sample were 0.0087–0.0151, 0.0021–0.0093 and 0.0037–0.0104, respectively. The ethanolicextract of K. alvarezii showed higher inhibitory effect than

Fig. 4. Inhibitory effect of K. alvarezii extract on the primary oxidation of a linoleic acid system, using the ferric thiocyanate method.

294 K.S. Kumar et al. / Food Chemistry 107 (2008) 289–295

did the positive control, BHT. This might be due to thepresence of ascorbic acid and vitamin A (b-carotene) con-tent in the extract of K. alvarezi (Fayaz et al., 2005).

Algal polysaccharides play an important role as free rad-ical-scavengers in vitro and antioxidants for the preventionof oxidative damage in living organisms. Their activitydepends on several structural parameters, such as the degreeof sulfation (DS), the molecular weight, the sulfation posi-tion, type of sugar and glycosidic branching. Moreover,some reports reveal that the sulfate and phosphate groupsin the polysaccharides lead to differences in their biologicalactivities. In vitro antioxidant activity of j-carrageenan oli-gosaccharides and their oversulfated, acetylated, and phos-phorylated derivatives have been reported by Yuan et al.(2005b). They also reported that phosphorylated and sul-fated glucans exhibited better antioxidant ability than didglucans or other neutral polysaccharides, which indicatesthat polyelectrolytes, such as glucan sulfate or phosphate,might have increased scavenging activity. Moreover, the sul-fate content of polysaccharides from Porphyra yezoensis

was reported to contribute to the antioxidant activity. Thecell wall of K. alvarezii is known to be constituted of carra-geenan, a sulfated polysaccharide, which may contribute toits antioxidant potential in addition to the presence of ascor-bic acid, vitamin A and various phenolics.

4. Conclusion

In the present investigation, the various solvent extractsof K. alvarezii exhibited excellent scavenging effect (%) byDPPH� assay, reducing power, ferrous ion-chelating activ-ity and antioxidant property in the linoleic acid system.Thus they could be used in nutraceutical and functionalfood applications. Since this is a preliminary study, adetailed investigation on the compositions of each compo-nent involved is absolutely necessary to establish appropri-

ate applications which may open new frontiers for humanconsumption of this seaweed world-wide.

Acknowledgements

The authors are grateful to Dr. Pushpito Ghosh, Direc-tor, Central Salt & Marine Chemicals Research Institute,Bhavnagar, Gujarat, India for his constant support andencouragement. They also appreciate Discipline Co-ordi-nator, Marine Biotechnology and Ecology Discipline, forproviding research facilities and profusely thank theDepartment of Biotechnology (Sanction No: BT/PR3309/PID/03/139/2002), New Delhi, India for providingfinancial assistance.

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Yuan, Y. V., Carrington, M. F., & Walsh, N. A. (2005a). Extracts fromdulse (Palmaria palmata) are effective antioxidants and inhibitors ofcell proliferation in vitro. Food Chemical Toxicology, 43,1073–1081.

Yuan, H., Zhang, W., Li, X., Lu, X., Li, N., Gao, X., et al. (2005b).Preparation and in vitro antioxidant activity of j-carrageenan oligo-saccharides and their oversulfated, acetylated, and phosphorylatedderivatives. Carbohydrate Research, 340, 685–692.

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Journal of Hazardous Materials 143 (2007) 590–592

Short communication

Phycoremediation of heavy metals by the three-color forms ofKappaphycus alvarezii

K. Suresh Kumar, K. Ganesan, P.V. Subba Rao ∗Marine Algae and Marine Environment Discipline, Central Salt and Marine Chemicals Research Institute,

Gijubhai Badheka Marg, Bhavangar 364 002, Gujarat, India

Received 16 June 2006; received in revised form 7 August 2006; accepted 20 September 2006Available online 26 September 2006

bstract

In the present investigation, three living color forms (brown, green and pale yellow) of Kappaphycus alvarezii were examined for their biosorp-ion ability in the laboratory. The brown color form proved to be an excellent metal biosorbent, i.e. it could adsorb good amount of cadmium.064 mg/100 g f.wt. and cobalt 3.365 mg/100 g f.wt. It also removed 2.799 mg/100 g f.wt. of chromium. The green color form absorbed 2.684,

.43 and 2.692 mg/100 g f.wt. of cadmium, cobalt and chromium, respectively. In contrast, the pale yellow form removed almost equal proportionf cadmium 0.961 mg/100 g f.wt. and chromium 0.942 mg/100 g f.wt. It also removed 1.403 mg/100 g f.wt. cobalt. Thus, the living color forms ofhis seaweed could form an effective biosorbent material for removal of heavy metals.

2006 Elsevier B.V. All rights reserved.

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eywords: Biosorption; Color forms; Heavy metals; Kappaphycus alvarezii; M

. Introduction

Metal sorption involves binding of metals onto the cell sur-ace and to intracellular ligands [1]. Metal remediation throughommon physico-chemical techniques is expensive and not eco-riendly. Hence, biotechnological approaches have received areat deal of attention as an alternative tool in the recent years.iosorption, the process of passive cation binding by dead or liv-

ng biomass, provides a potentially cost-effective way of remov-ng toxic metals from industrial wastewaters [2], and it coulde employed most effectively in a concentration range below00 mg l−1, where other techniques are ineffective or costly3,4]. Metal ion binding during biosorption processes has beenound to involve complex mechanism, such as ion-exchange,omplexation, electrostatic attraction and microprecipitation5]. There have been some indications that ion-exchange playsn important role in metal sorption by algal biomass [6].

pplicability of growing bacterial/fungal/algal cells for metal

emoval and the efforts directed towards cell/process develop-ent to make this option technically and economically viable

∗ Corresponding author. Tel.: +91 278 2568694; fax: +91 278 2566970.E-mail address: pvsubbarao@csmcri.org (P.V. Subba Rao).

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304-3894/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.jhazmat.2006.09.061

or the treatment of metal rich effluents have been reviewed byalik [7].Marine algae (seaweeds) are readily available in large quanti-

ies for the development of highly effective biosorbent materials.owever, considering the large number of macro-algal species

dentified so far, only a few have been studied for their heavyetal uptake properties. Most of these studies are limited to thescophyllum and Sargassum sp. [8]. The non-living biomass ofarine algae Sargassum sp., Padina sp., Ulva sp., and Gracil-

aria sp., have been investigated for their biosorption perfor-ance in the removal of lead, copper, cadmium, zinc, and nickel

rom dilute aqueous solutions. It was also found that the biosorp-ion capacities were significantly affected by solution pH, withigher pH favoring higher metal-ion removal [9]. A number oforkers investigated the feasibility of using cheaply availablearine or fresh water algae for heavy metal removal [10,11].he passive removal of toxic heavy metals by brown marinelgae via biosorption was reported by Davis et al. [12,13], whottributed this property to cell wall polysaccharides like alginatend fucoidan. On the other hand, high adsorption capacities of

arious low cost adsorbents, e.g. chitosan (815, 273, 250, 222,5 mg/g of Hg2+, Cr6+, Cd2+, Cu2+, and Zn2+, respectively),eolites (175 and 137 mg/g of Pb2+ and Cd2+, respectively),aste slurry (1030, 560, 640 mg/g of Pb2+, Hg2+, and Cr6+,

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Kmft0tively. Similarly, the green color form showed a biosorption of2.684, 3.430 and 2.692 mg/100 g f.wt. of cadmium, cobalt andchromium respectively. However, the brown color form is thesuperior one in biosorption of cadmium and cobalt followed by

Table 1Biosorption (mg/100 g f.wt.) of heavy metals by three-color forms of Kappa-phycus alvarezii

Color form Metal Control Biosorption

Brown Cd 0.0225 3.064Co 0.0060 3.365Cr 0.0040 2.799

Green Cd 0.0050 2.684Co 0.0040 3.430

K. Suresh Kumar et al. / Journal of H

espectively), and lignin (1865 and 95 mg/g of Pb2+ and Zn2+,espectively) have been reported [14]. Removal of zinc fromqueous solutions using bagasse fly ash, a waste from sugar canendustry as a low cost adsorbent has also been studied by Guptand Sharma [15]. Fresh algal biomass of Spirogyra species wassed as biosorbent for the removal of Cr(VI) from aqueous solu-ions [16].

Kappaphycus alvarezii [17] is a promising carrageenophytend three-color forms (brown, red and green) have been reportedn this alga from Philippines [18]. In India, three-color formsbrown, green and pale yellow) were also obtained during theultivation of this alga at Port Okha, northwest coast of India.his study is aimed at using these living color forms of thiseaweed in the laboratory for biosorption of toxic metals liked, Co and Cr.

. Materials and methods

.1. Collection of samples

Three-color forms (brown, green and pale yellow) of Kap-aphycus alvarezii were obtained at Port Okha (22◦28′N and9◦05′E) Northwest coast of India during cultivation (April006). The collected fresh algae were thoroughly washed withterilized seawater to eliminate the adhering foreign materials,uch as sand and debris, and materials used for heavy metalptake studies.

.2. Heavy metal uptake

The following sets of experiments for biosorption stud-es were designed. Three 500 ml flat bottom flask with spoutor air circulation were used containing 400 ml of sterilizedeawater to which 25 mg l−1 each of cadmium, cobalt andhromium, as cadmium sulfate, cobalt sulfate and potassiumichromate (analytical grade) was added (pH 7.7). To avoideavy metal contamination, the glassware was soaked in 10%NO3 for 24 h, rinsed with deionised water and oven dried prior

o use.Ten grams of each living algal color form (a single thallus

ragment) was added aseptically to the three respective flasksontaining mixture of heavy metals. One set without heavy metalas also inoculated with each color form that served as control.xperiments were conducted in duplicate.

The living color forms were maintained at 25 ◦C ± 1, in alean environment where aeration was continuously provided.fter 5 days of incubation, the algae were removed and shaderied at room temperature followed by drying in an oven at0 ◦C for 1 h in a porcelain crucible. These samples were ashedt 550 ◦C in muffle furnace for 2 h. The ash was cooled at roomemperature, moistened with 10 drops of distilled water and care-ully dissolved in 3 ml HNO3 (1:1 v/v). The crucible was then

eated on a hot plate at 110 ◦C till the acid solution totally evap-rated. The crucible was returned to muffle furnace and ashedgain for 1 h at 550 ◦C and cooled. Subsequently the ash wasissolved in 10 ml of HCl (1:1 v/v), and the solution was filtered

P

ous Materials 143 (2007) 590–592 591

hrough millipore filter paper (0.25 �) into a 50 ml volumetricask and 2 ml 0.1N HNO3 was added to the filtrate and the finalolume was made up to 50 ml using distilled water [19]. Thisas subjected to heavy metal analysis.

.3. Instrumentation

Analysis of heavy metals cadmium (Cd), cobalt (Co) andhromim (Cr) was carried out using inductively coupled plasmatomic emission spectroscopy, ICP-AES (Perkin-Elmer, Optima000). The mean value of the results obtained here was consid-red and the heavy metal uptake was calculated as mg/100 g f.wt.

. Results and discussion

The three-color forms of Kappaphycus alvarezii were com-ared for their metal biosorption efficiency, as well as survivalnder stressed condition (i.e. presence of different heavy metal)fter 5 days of incubation. It was observed that all the three-olor forms could survive in this stress environment. This coulde considered as a positive indication for this seaweed to be usedor phycoremediation.

Terry and Stone [20] reported that living algae are knowno adsorb more heavy metals due to metabolic uptake andontinuous growth, e.g. cadmium and copper biosorption bycenedesmus abundans. Sloof et al. [21] found that cadmiumptake by living Selenastrum capricornutum was rapid in therst adsorption stage, and then continued more slowly in thehysiological metabolic stage. Resistance, accumulation andllocation of zinc in two ecotypes of the green alga Stigeoclo-ium tenue Kutz coming from different habitats with differenteavy metal concentrations has been reported [22].

As seen in Table 1, heavy metal treated brown color form ofappaphycus alvarezii could sorb maximum metals, i.e. cad-ium, cobalt and chromium 3.064, 3.365 and 2.799 mg/100

resh weight (f.wt.) respectively. The pale yellow color formreated with metal could take up only 0.961, 1.403 and.942 mg/100 g f.wt. of cadmium, cobalt and chromium respec-

Cr 0.0045 2.692

ale yellow Cd 0.0310 0.961Co 0.0160 1.403Cr 0.0230 0.942

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92 K. Suresh Kumar et al. / Journal of H

reen and pale yellow color forms of this seaweed. Biosorp-ion of cobalt in the brown color form is slightly less than thereen one. The control contained very little amount of heavyetal reflecting that these are absorbed from the surrounding

nvironment (seawater) where they are cultivated.It is observed that all the three color forms of Kappaphy-

us alvarezii exhibited high cadmium, cobalt and chromiumdsorption capacities when tested as fresh material. This is ofxtreme significance, as most algae do not survive in stressednvironment containing heavy metals. The algae could not onlyurvive up to a period of 5 days but also retained its uniquebility to remove heavy metal ions. High adsorption efficiencyf the algae, low biomass cost (mainly transportation cost), lessabor input and high yields of biomass under cultivation makeshis process of biosorption an effective, cheap and alternativeechnique for treatment of metal-bearing polluted marine envi-onment.

Biosorption of cadmium and copper contaminated water bycenedesmus abundans revealed that living algae could reduceadmium from 10 to 0.10 mg/l in 36 h [20]. In the presenttudy the brown color form of Kappaphycus alvarezii provedompetent enough as it could adsorb 3.064 mg/100 g f.wt.admium.

Biosorption efficiency of brown algae, Macrocystis pyrifera,jellmaniella crassiforia and Undaria pinnatifida have beenxploited for the recovery of lead and cadmium ions [23]. Itas been reported that alkali-pretreated Ulva biomass showedhe sorption capacity (qm) from 60 to 90 mg g−1 and the sorp-ion affinity from 0.03 to 0.04 l mg−1 at pH 7.8 while studyinghe uptake of cadmium, copper and zinc [24].

Studies on time dependent heavy metal uptake (by increasingr decreasing the contact time) could be taken up based on theositive results obtained here. Moreover, heavy metal uptakey biomass of these three-color forms would surely prove thefficiency of this seaweed as an effective biosorbent.

Cultivation and use of this seaweed as a biosorbent in differentarts of the world could be embarked upon as a greener (envi-onment friendly) and profitable approach leading to employ-ent generation for coastal living people on one hand and

leaning the environment on the other hand. This is an eco-riendly option for remediation of various coastal waters andould help develop a new scope of research. Employing this

eaweed for biosorption studies fulfils the parameters like eco-riendliness and economic feasibility as suggested by Mehta andaur [1].

cknowledgments

The authors are grateful to Dr. Pushpito K. Ghosh, Director,entral Salt & Marine Chemicals Research Institute, Bhav-agar, Gujarat, India for his constant support and encourage-ent. They also appreciate the facilities provided by Discipline

o-ordinator, Marine Algae & Marine Environment Disciplinend profusely thank the Department of Biotechnology, Newelhi, India for providing financial assistance (sanction no.:T/PR3309/PID/03/139/2002).

[

ous Materials 143 (2007) 590–592

eferences

[1] S.K. Mehta, J.P. Gaur, Use of algae for removing heavy metal ions fromwastewater: progress and prospects, Crit. Rev. Biotechnol. 25 (2005)113–152.

[2] N. Kuyucak, B. Volesky, Biosorption by algal biomass, in: B. Volesky (Ed.),Biosorption of Heavy Metals, CRC Press, Boca Raton, Florida, USA, 1990,p. 175.

[3] R.H.S.F. Vieira, B. Volesky, Biosorption: a solution to pollution? Int. Micro-biol. 3 (2000) 17–24.

[4] S. Schiewer, B. Volesky, Modelling of the proton-metal ion exchange inbiosorption, Environ. Sci. Technol. 29 (1995) 3049–3058.

[5] B. Volesky, Z.R. Holan, Biosorption of heavy metals, Biotechnol. Prog. 11(1995) 235–250.

[6] B. Volesky, M.M. Figueira, V.S. Ciminelli, F.A. Roddick, Biosorption ofmetals in brown seaweed biomass, Water Res. 34 (2000) 196–204.

[7] A. Malik, Metal bioremediation through growing cells, Environ. Int. 30(2004) 261–278.

[8] M.Q. Yu, J.T. Matheickal, P. Yin, P. Kaewsarn, Heavy metal uptakecapacities of common marine macroalgal biomass, Water Res. 33 (1999)1534–1537.

[9] P.X. Sheng, Y.P. Ting, P.J. Chen, L. Hong, Sorption of lead, copper,cadmium, zinc, and nickel by marine algal biomass: characterization ofbiosorptive capacity and investigation of mechanisms, J. Colloids Interf.Sci. 275 (2004) 131–141.

10] D.W. Darnall, B. Greene, M. Hosea, R.A. Mcpherson, M. Henzl, M.D.Alexander, Recovery of heavy metals by immobilized algae, in: R. Thomp-son (Ed.), Trace Metal Removal from Aqueous Solutions, The RoyalSociety of Chemistry, London, 1986, pp. 1–25.

11] Z.R. Holan, B. Volesky, I. Prasetyo, Biosorption of cadmium by biomassof marine algae, Biotechnol. Bioeng. 41 (1993) 819–825.

12] T.A. Davis, B. Volesky, M. Alfonso, A review of the biochemistry of heavymetal biosorption by brown algae, Water Res. 37 (2003) 4311–4330.

13] T.A. Davis, F. Llanes, B. Volesky, A. Mucci, Metal selectivity of Sargassumspp. and their alginates in relation to their l-guluronic acid content andconformation, Environ. Sci. Technol. 37 (2003) 261–267.

14] S. Babel, T.A. Kurniawan, Low-cost adsorbents for heavy metals uptakefrom contaminated water: a review, J. Hazard. Mater. B 97 (2003) 219–243.

15] V.K. Gupta, S. Sharma, Removal of zinc from aqueous solutions usingbagasse fly ash—a low cost adsorbent, Ind. Eng. Chem. Res. 42 (25) (2003)6619–6624.

16] V.K. Gupta, A.K. Srivastava, N. Jain, Biosorption of chromium(VI) fromaqueous solutions by green algae spirogyra species, Water Res. 35 (2001)4079–4085.

17] E.I. Ask, R.V. Azanza, Advances in cultivation technology of commer-cial eucheumatoid species: a review with suggestions for future research,Aquaculture 206 (2002) 257–277.

18] A.Q. Hurtado-Ponce, Carrageenan properties and proximate composi-tion of three morphotypes of Kappaphycus alvarezii Doty (Gigartinales,Rhodophyta) grown at two depths, Bot. Mar. 38 (1995) 215–219.

19] J.B. Jones, Plants, in: S. William (Ed.), An Official Method of Analysis,Association Official Analytical Chemists, Arlington, VA, USA, 1984, pp.38–64.

20] P.A. Terry, W. Stone, Biosorption of cadmium and copper contaminatedwater by Scenedesmus abundans, Chemosphere 47 (2002) 249–255.

21] J.E. Sloof, A. Viragh, B. Van Der Veer, Kinetics of cadmium uptake bygreen algae, Water Air Soil Pollut. 83 (1995) 105–122.

22] B. Pawlik-Skowronska, Resistance accumulation and allocation of zinc intwo ecotypes of the green alga Stigeoclonium tenue Kutz coming fromhabitats of different heavy metal concentrations, Aquat. Bot. 75 (2003)189–198.

23] H. Seki, S. Akira, Biosorption of heavy metal ions to brown algae, Macro-

cystis pyrifera, Kjellmaniella crassiforia, and Undaria pinnatifida, J. Col-loids Interf. Sci. 206 (1998) 297–301.

24] Y. Suzuki, T. Kametani, T. Maruyama, Removal of heavy metals fromaqueous solution by nonliving Ulva seaweed as biosorbent, Water Res. 39(2005) 1803–1808.

Heavy metal chelation by non-living biomass of three colorforms of Kappaphycus alvarezii (Doty) Doty

K. Suresh Kumar & K. Ganesan & P. V. Subba Rao

Received: 18 April 2007 /Accepted: 26 April 2007 / Published online: 15 June 2007# Springer Science + Business Media B.V. 2007

Abstract Water pollution by toxic heavy metals is aburning environmental problem and has presented achallenge to humans. Removal of heavy metals usingnon-living biomass of seaweeds could be a potentialsolution to this problem. In the present investigation,biomass of three color forms of Kappaphycus alvarezii,viz. brown, green and pale yellow, were studied in thelaboratory for their heavy metal chelating capacity usingcadmium, cobalt, chromium and copper. Amongst the fourconcentrations used (25, 50, 75 and 100 mg L−1) maximumchelation of Cd, Co and Cu was recorded at 25 mg L−1

concentration. The highest amount of Cr was chelated at100 mg L−1 by all the three color forms. The pale yellowform showed maximum chelation for all four metalsstudied. Further, chelation in all the color forms was foundto be: Cd 5.37±0.59–15.84±0.32 %, Co 21.19±0.13–32.32±0.62 %, Cr 65.38±0.27–88.09±0.51 % and Cu 59.53±0.37–90.28±0.89 %. All the three color forms of K.alvarezii serve as an excellent biodetoxifier as they allchelated considerable amounts of heavy metals.

Keywords Biosorbent . Chelation . Color forms .

Heavymetals .Kappaphycus alvarezii

Introduction

Heavy metal pollution is an environmental problem ofworldwide concern. Some industrial processes result in the

release of heavy metals into natural water systems, leadingto increasing concern about their toxic effect as environ-mental contaminants. Lead (Pb), copper (Cu), cadmium(Cd), zinc (Zn) and nickel (Ni) are among the mostcommon pollutants found in industrial effluents. Even atlow concentrations these metals can be toxic to livingorganisms, including humans. Conventional methods forremoving heavy metals (precipitation, chemical oxidation/reduction, ion-exchange, reverse-osmosis, membrane sepa-ration, etc.) are often ineffective and costly (Marques et al.1991; Volesky 1994; Dey et al. 1995; Kapoor andViraraghavan 1995; Lu and Wilkins 1996; Zhao andDuncan 1997). Recently, biological removal processes forremoving heavy metals from aqueous wastes have beenattracting considerable attention. These include newapproaches such as the use of marine algal biomass forbiosorption of heavy metals (Fourest and Roux 1992;Mattuschka and Straube 1993; Volesky 1994; Volesky andHolan 1995). Algae are of special interest in the search for,and development of, new biosorbent materials due to theirhigh sorption uptake and their ready availability inpractically unlimited quantities in the seas and oceans(Feng and Aldrich 2004). Biosorption properties of a fewalgae are accredited to their cell-wall polysaccharides likealginate and fucoidan, which have a high affinity fordivalent cations (Fourest et al. 1994; Puranik et al. 1999;Khoo and Ting 2001; Chen et al. 2002; Davis et al. 2003).The non-living biomass of Sargassum species, Macrocystispyrifera, Kjellmaniella crassiforia, Undaria pinnatifida andUlva species are known to effectively remove Cd, Cu, Zn,Cr and Ni (Seki and Suzuki 1998; Yang and Volesky 1999;Davis et al. 2000; Suzuki et al. 2005). Kappaphycusalvarezii, a potential carageenophyte, is reported to occurin brown, green, pale yellow and red color forms in fieldcultivation (Dawes 1992; Hurtado-Ponce 1995; Suresh

J Appl Phycol (2008) 20:63–66DOI 10.1007/s10811-007-9181-8

K. Suresh Kumar :K. Ganesan : P. V. Subba Rao (*)Marine Biotechnology and Ecology Discipline,Central Salt and Marine Chemicals Research Institute,Bhavangar,364 002 Gujarat, Indiae-mail: pvsubbarao@csmcri.org

Kumar et al. 2007). In an earlier study the three living colorforms (brown, green and pale yellow) of K. alvarezii wereexamined in the laboratory for their biosorption ability touptake Cd, Co and Cr (Suresh Kumar et al. 2007). Thepresent work is aimed at studying the chelation of Cd, Co,Cr and Cu from aqueous solution by the brown, green andpale yellow color forms of non-living biomass of K.alvarezii in the laboratory.

Materials and methods

The three color forms—brown, green and pale yellow—ofKappphycus alvarezii (Doty) Doty were obtained in April2006 from the cultivation farm of Port Okha (L 22°28.528′ N;L 069° 04.322′ E), on the northwest coast of India.

Brown color form: Plants are dark brown in color, branchesare thick and robust, profusely and irregularly branched, thebranches are tapering towards the tips. The basal part ofthe branch is very thick; plant normally measures up to20–25 cm.

Green color form: Plants are dark green in color, branchesare thick and profusely branched with fragile tips; plants aresmaller than the brown form and measure up to 20 cm.

Pale yellow form: Plants are light yellowish in color,smaller than the other two color forms, less branched.Branches very fragile and break even at moderate watercurrent; the plant measures up to 14–16 cm.

The collected samples were thoroughly washed withseawater to remove epiphytes and dirt particles and thendried in the shade for 2 days. Samples were then brought tothe laboratory and oven-dried at 80°C for 3 h to constantweight before being pulverized (size 2 mm) in a grinder.This non-living biomass was used for further experiments.

Heavy metal chelation

Chelation of heavy metals was studied by adding a 1 gsample of each color form to an Erlenmeyer flaskcontaining 100 mL aqueous solution of Cd, Co, Cr andCu at four concentrations, i.e., 25, 50, 75 and 100 mg L−1

prepared using analytical grade cadmium sulfate, cobaltsulfate, potassium dichromate and copper sulfate. Controlsfor each color form without addition of heavy metals werealso maintained. The pH was adjusted initially to 4.5 andwas found to be 5.3 at the conclusion of experiment. Allexperiments were conducted in triplicate. To avoid heavymetal contamination, glassware was soaked in 10% HNO3

for 24 h and rinsed with deionized water prior to use. Flaskswere incubated for 72 h on a shaker at room temperature(33±1°C) and the flask contents were subsequently syringe

filtered (0.22 μm pore size; Millipore) and subjected tofurther analysis.

Instrumentation

Heavy metal content in all the filtrates was quantified usinginductively coupled plasma optical emission spectroscopy(ICP-OES; Perkin Elmer Optima-2000 DV). The differencebetween the amount of metal present in the filtrate and thetotal amount of metal present initially in the flask yieldedthe amount of metal chelated by the biomass. Thepercentage chelation for each metal was estimated. Meansand standard deviations were calculated.

a

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40

60

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100

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Metal concentrations (ppm)

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Fig. 1a–c Heavy metal chelation by three color forms of Kappphycusalvarezii. a Brown, b green, c pale yellow

64 J Appl Phycol (2008) 20:63–66

Results and discussion

The chelating efficiency of the non-living biomass of thethree color forms of K. alvarezii (Fig. 1) revealed that themaximum amount of Cd, Co and Cu were chelated (%)when the heavy metal concentration was 25 mg L−1, i.e.,the brown form chelated Cd 5.37±0.59, Co 21.19±0.13,Cu 59.53±0.37; the green form chelated Cd 8.87±0.61, Co28.54±0.13, Cu 70.87±0.50; and the pale yellow formchelated Cd 15.84±0.32 , Co 32.32±0.62 , Cu 90.28±0.89.In the case of Cr, maximum chelation was observed at100 mg L−1 concentration, i.e. brown 68.79±0.20, green77.21±0.42 and pale yellow 88.10±0.15.

Chelation decreased with increasing concentration of Cd,Co and Cu, but increased with increasing concentrations ofCr. Furthermore, at 100 mg L−1 concentration, no chelationof Cd or Co was found in the brown color form, and nochelation of Cd in the green color form. Of the four metalsused, the highest chelation was recorded with Cr followedby Cu, Co and Cd in the brown and green forms. However,a different trend was observed in the pale yellow form,where maximum chelation of Cu was noted, followed byCr, Co and Cd. This color form chelated highest amount ofheavy metals, followed by the green and brown forms. Thepale yellow form of K. alvarezii is known to chelate 65.28±0.51–90.28±0.89 % of copper and 80.86±0.86–88.10±0.15 % of chromium. Lee et al. (2000) demonstrated 57%chromate chelation by a red algae Pachymeniopsis species.A brown seaweed, Padina species, is known to chelatecopper (0.80 mmolg-1; Kaewsarn, 2002). Dried biomass ofSargassum wightii is also reported to chelate maximallycadmium metal followed by lead, copper and zinc,indicating the affinity range for heavy metal ions (Kumarand Kaladharan 2006). Brown seaweed biomass of Ecklo-nia maxima, Macrocystis angustifolia and Laminariapallida are known to sequester copper, zinc and cadmiumions at concentrations likely to be encountered in wastewater, i.e., 0–100 mg L−1 (Stirk and Van Staden 2000).Ascophyllum nodosum out-performed a commercial ionexchange resin (DUOLITE GT-73) by accruing 100 mgCd2+/g biomass (Holan et al 1993). Hashim and Chu (2004)and Tsui et al. (2006) have observed that brown seaweedsexhibit better metal chelation properties than their redcounterparts. In contrast, in the present study, the redseaweed K. alvarezii has demonstrated more chelation ofheavy metals. In particular, a notable increase in chelationof Cr (68.80–88.08%) was recorded by increasing theconcentration of Cr in the aqueous solution.

Conclusion

The non-living biomass of all three color forms of K.alvarezii, a carageenan yielding red seaweed, proved to be

an efficient biosorbent material, especially for chelation ofCr and Cu from aqueous solution. This is in contrast withresults obtained by Vieira and Volesky (2000), who opinedthat red marine algae containing carageenan do not haveoutstanding metal-sorbing properties despite having poten-tial binding sites.

The greater chelation of heavy metals by the pale yellowform of K. alvarezii in comparison with that by the othertwo forms (brown and green) might be attributed to thecumulative inherent physiological characteristics, especial-ly those related to accumulation of pigments and cara-geenan at specific binding sites. Since the experimentsconducted here were preliminary in nature, further study isvery much necessary to understand the mechanism andkinetics of uptake of heavy metals, which may broaden thescope to use K .alvarezii color forms for bioremediation.However, the present investigation shows that all threecolor forms of K. alvarezii exhibit excellent heavy metalchelating capacity, thus proving them to be potentialbiodetoxifiers.

Acknowledgments The authors are grateful to Dr. Pushpito Ghosh,Director, Central Salt and Marine Chemicals Research Institute,Bhavnagar, Gujarat, India, for his constant support and encourage-ment. They also appreciate the Discipline Co-ordinator of the MarineBiotechnology and Ecology Discipline, for providing researchfacilities, and profusely thank the Department of Biotechnology(Sanction No: BT/ PR 3309 / PID / 03 / 139 / 2002), New Delhi,India for providing financial assistance.

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