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Biosorption of Chromium, Cadmium, and Cobalt from Aqueous Solution byImmobilized Living Cells of Chryseomonas luteola TEM 05enay Hamarat Baysal a; Seçil Önal a; Güven Özdemir b

a Biochemistry Department, Faculty of Science, Ege University, Bornova-zmir, Turkey b Biology Department,Faculty of Science, Ege University, Bornova-zmir, Turkey

Online Publication Date: 01 October 2009

To cite this Article Baysal, enay Hamarat, Önal, Seçil and Özdemir, Güven(2009)'Biosorption of Chromium, Cadmium, and Cobalt fromAqueous Solution by Immobilized Living Cells of Chryseomonas luteola TEM 05',Preparative Biochemistry andBiotechnology,39:4,419 — 428

To link to this Article: DOI: 10.1080/10826060903209794

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Biosorption of Chromium, Cadmium, and Cobaltfrom Aqueous Solution by Immobilized

Living Cells of Chryseomonas luteola TEM 05

Senay Hamarat Baysal,1 Secil Onal,1 and Guven Ozdemir2

1Biochemistry Department, Faculty of Science, Ege University,Bornova-Izmir, Turkey

2Biology Department, Faculty of Science, Ege University,Bornova-Izmir, Turkey

Abstract: In this work, the potential use of the immobilized cells of Chry-seomonas luteola TEM 05 for the removal of Crþ6, Cdþ2 and Coþ2 ions fromaqueous solutions was investigated. The living cells of C. luteola TEM 05 werefirstly entrapped both in carrageenan and chitosan coated carrageenan gelsand then used in biosoption of the metal ions in batch reactors at pH 6.0,25�C, in 100mgL�1 of each metal solution. Besides this, a process of com-petitive biosorption of these metal ions was also described and compared tosingle metal ion adsorption in solution. According to the immobilizationresults, the replacement of KCl by KCl-chitosan as gelling agent improvedthe mechanical strength and thermal stability of the gel. In addition, the C.luteola TEM 05 immobilized carrageenan-chitosan gel system was quite moreefficient for the fast adsorption of metal ions from aqueous solution thanthe carrageenan gels without biomass.

Keywords: Biosorption, Carrageenan, Chitosan, Chryseomonas luteola, Heavymetal

Correspondence: Assoc. Prof. Dr. Secil Onal, Biochemistry Department,Faculty of Science, Ege University, 35100 Bornova-Izmir, Turkey. E-mail: secil.onal@ege.edu.tr

Preparative Biochemistry & Biotechnology, 39: 419–428, 2009

Copyright # Taylor & Francis Group, LLC

ISSN: 1082-6068 print/1532-2297 online

DOI: 10.1080/10826060903209794

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INTRODUCTION

The increase in industrial activities has intensified environmentalpollution and the deterioration of some ecosystems, with the accumula-tion of pollutants such as heavy metals, synthetic compounds, wastenuclear liquids, etc. Growing attention is being given to the potentialhealth hazard presented by heavy metals to the environment.[1] Heavymetal ions are used in various industries due to their technologicalimportance. Wastewaters from these industries include metal ions havingpermanent toxic effects. The toxicity of heavy metals may be caused bymechanisms that include blocking essential functional groups of bio-molecules and disrupting the integrity of bioadsorbents.[2] The metalsof most immediate concern are Cr, Mn, Fe, Cu, Zn, Hg, Pb, and Cd.These metals are widely distributed in materials, which make up theearth’s surface. For humans, poisoning by most of these metals causessevere dysfunction of the kidney, reproductive system, liver, brain, andcentral nervous system.[3–5]

Removal and recovery of heavy metals are very important withrespect to environmental and economical considerations. The traditionalmethods (chemical precipitation and filtration, electrochemicaltreatments, reverse osmosis, ion exchange, adsorption, and evaporation)for the removal of heavy metals from water are generally expensive orinadequate to treat highly dilute solutions.[6] The use of biological materi-als for removing and recovering heavy metals has emerged as a potentialalternative method to conventional techniques, which may be expensiveor ineffective. The uptake of metals by free or immobilized micro-organisms from solutions, including two or more metal ions, has gainedimportance recently. This is known as biosorption, which is passive inthat no energy is required. Algae, bacteria, fungi, and yeasts have provedto be potential metal sorbents. Investigations on the biosorption mechan-ism of heavy metals show that the metal ions are deposited by adsorptionto the functional groups presented on the cell wall.[6–8]

Immobilized cell systems have the potential to degrade toxicchemicals faster than conventional wastewater treatment systems, sincehigh densities of specialized microorganisms are used in immobilized cellsystems. One of the most widely used techniques for cell immobilizationis cell entrapment, in which the microbial cells are enclosed in a polymericmatrix which is porous enough to allow the diffusion of substrate to cellsand product away from the cells. Polysaccharide gel immobilizedmicroorganisms can be used to remove heavy metal ions from aqueoussolutions, providing an alternative to physicochemical technologies forwastewater treatment.[9–11] The carrageenans constitute a class of linear,sulfated polysaccharides extracted from various species of theRhodophyta (marine red alga). k-Carrageenan consists of alternating

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structures of D-galactose 4-sulphate and 3,6-anhydro-D-galactose 2sulfate.[12] Chitosan, also named poly (b-1,4)-2-amino-2-deoxy-D-glucopyranose, a poly (D-glucosamine) is prepared from chitin bydeacetylating its amido groups.[13–16] Polyelectrolyte complexes, formedby mixing polysaccharides of opposite charge, have recently attractedconsiderable attention because of their potential in various biotechnolo-gical applications.[11,17]

In this study, biosorption of each of the Crþ6, Cdþ2, and Coþ2 ionsby a floc forming bacterium, C. luteola TEM 05, naturally present inactivated sludge, entrapped in carrageenan was investigated and partialcompetitive adsorptions of multi-metal ions were also investigated.

EXPERIMENTAL

Chemicals

All chemicals used in this study were analytical grade and were purchasedfrom Sigma Chem. Co. (St. Louis, MO) and Fluka Chemie AG (Buchs,Switzerland). Chitosan was provided from Marine Chemicals (India).

Isolation and Preparation of Microorganism

The employed procedure for the isolation of floc-formers was a modifi-cation of that used by and formulated to enhance slime production(ESP).[18] Purified isolates were maintained on a chemically definedmedium solidified by addition of Bacto No 1 agar (2% w=v). To isolatethe most exocellular polysaccharide (EPS) producing bacterium, samplesfrom wastewater treatment of a petrochemical industry (Izmir, Turkey)were passed aseptically through ten-fold dilutions of Ringers solutionto yield final concentrations of 10�4 and 10�5. Spread plates were pre-pared from each dilution (0.1mL) on ESP medium (pH 7.0) with 8.5%of glucose and incubated at 30�C for 3 days. Discrete colonies werepicked from each plate into Erlenmayer flasks (250mL) containingESP medium (100mL) and grown at 30�C with gyratory shaking(200 rev min�1) for 5 days. The contents of each flask were then decantedinto 3 volumes of propan-2-ol, shaken vigorously, and held at 4�C for 4 h.The most EPS producing organism was selected for this study.

A pure colony of the strain TEM 05 was identified presumptively onthe following features: colony morphology, colonial pigmentation, cellmorphology, gram-staining reaction, oxidase positivity. Isolates werefurther characterized biochemically using the API 20E and 20 NE Kits(Analytical Profile Index, Bio Merieux SA, France). The MICs of metals

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were determined with microtiter plates containing nutrient broth andincreasing amounts (0–3000 mg=mL) of either metal.[19,20]

In this study, C. luteola, which was isolated from activated sludgeflocs wastewater treatment a petrochemical industry, was cultivatedaerobically in 500mL conical flasks containing sterile nutrient broth(Difco) on a rotary shaker (100 rev min�1) at 30�C. Cells were harvestedat the end of the exponential phase, i.e., after 48 h incubation. Aftercultivation, the cells were centrifuged at 10,000 g for 20min and then usedfor biosorption studies.

Immobilization of C. luteola TEM 05 in Carrageenan Gels

Two percent of natural carrageenan powder was dissolved in distilledwater by heating to 60�C and sterilized by autoclaving (121�C, 20min).After cooling to 40–45�C, it was mixed with cell suspension (10mL,about 1� 109 cellsmL�1) at the same temperature. In sterile conditions,the mixture was subsequently dropped into 2% KCl solution. The beadswere hardened in this solution for 2–6 h. The particles were washed withsterile physiological saline to remove excess potassium ions anduntrapped cells. The second procedure is the same as described above,except that 2% KCl solution was replaced with a mixture solution of2% KCl–1% chitosan.

The particle sizes of the beads were determined with the help of amagnifying glass. This was roughly checked by suspending the randomlychosen beads in water contained in a 10mL of graduated cylinder and bymeasuring the increase in volume. The procedure was repeated threetimes. The particle diameters were 3.30mm; 3.37mm; 3.43mm;3.31mm for carrageenan, carrageenan-biomass, carrageenan-chitosan,and carrageenan-chitosan-biomass, respectively.

Biosorption Studies

The biosorption of Crþ6, Cdþ2, and Coþ2 on the immobilized C. luteolaTEM 05 from artificial wastewaters containing single metal ions andmulti-metal ions were investigated in batch biosorption experiments.Each heavy metal ion (100mgL�1) was prepared in 50mL growthmedium and contacted with the beads (20 beads) in Erlenmeyer flasksagitated by an orbital shaker at 200 rpm. All biosorption experimentswere carried out at 25� 2�C. Liquid samples (0.1mL) were taken fromthe batch reactor at predetermined time intervals (every few minutes toevery few hours) and were analyzed to determine the residual metalconcentration in the aqueous solution. Blank experiments were run

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concurrently in all experiments. Each experiment was repeated threetimes and the results given are the average values. The amount of metalions adsorbed per unit empty or organism immobilized carrageenanpreparations were obtained by using the following expression:

Q ¼ ½ðC0 � CeÞ � V�=m

where C0 and Ce are the initial and final metal ion concentration (mg L�1),respectively. V is the volume of the medium (mL), and m is the amount ofthe biomass (g).

Analysis of Metal Ions

The concentration of unadsorbed metal ions the biosorption mediumwere determined by using a ICP-AES (model DRE, Leeman Labs Inc).A blank experiment was repeated three times and the results given arethe average values.

RESULTS AND DISCUSSION

The use of immobilized microorganisms has been gaining importance inthe last few decades. Immobilization of the biomass in solid structurescreates a material with the right size, mechanical strength, rigidity, andporosity necessary for use in unit operations typical of chemical engineer-ing. Various techniques are used for the biomass immobilization. Theprincipal techniques found in the literature for the application of biosorp-tion are based on adsorption on inert supports, on entrapment in apolymeric matrix, on covalent bonds to vector compounds, or on cellscross-linking. One of the most widely used techniques for cell immobiliza-tion is cell entrapment in natural gels such as carrageenan.[1,12,20]

Potassium chloride is commonly used as the gelling agent fork-carrageenan immobilized enzyme and cells. However, the mechanicalstrength of the gels is relatively low. We investigated the replacementof KCl by KCl þ chitosan as the gelling agent to improve the strengthof k-carrageenan gels. Chitosan is a polycationic polymer obtainedcommercially by alkaline deacetylation of chitin from shellfish exoskele-tons. Since it is inexpensive, non-toxic, and possesses potentially reactiveamino functional groups, chitosan has been shown to be of potential usein many different fields, including as a support for enzyme or micro-organism immobilization.[1,10,12] Chitosan forms polyelectrolytecomplexes with polyanionic polymers. Encapsulating processes basedon the electrostatic interaction between sodium alginate or carrageenan

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and chitosan resulting in the formation of a reinforced gel has beenreported.[10,20,21] According to our experience, through the addition ofchitosan into the gelling agent, the mechanical strength and thermal sta-bility of carrageenan gel were greatly improved. This was attributed tothe treatment of carrageenan gels with this highly charged polycation,forming a polyelectric complex which coats the surface of the beads.Chitosan, complexing with the polyanionic carrageenan reinforces thegel network through many points.[10,17]

Based on light-microscopic observation, C. luteola TEM 05, whichwas isolated from activated sludge flocks, is Gram-negative, motile,rod-shaped, and 1.0–1.8 mm in size. According to physiological character-ization results, the strain TEM 05 was identified as C. luteola.[22] The cells

Figure 1. Single and partial competitive adsorption of Crþ6, Cdþ2, and Coþ2 oncarrageenan (a) and C. luteola TEM 05 immobilized in carrageenan gels (b). [^;Co-Mix, &; Cr-Mix, ~; Cd-Mix, �; Co, &; Cr, D; Cd].

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were cultivated in nutrient broth media amended with various concentra-tions of Crþ6, Cdþ2, and Coþ2. It appears that the cells were able tosurvive metal concentrations as high as 125mgL�1 for Crþ6, 25mgL�1

for Cdþ2, and 180mgL�1 for Coþ2. Although MIC values of Cdþ2 ionsfor C. luteola TEM 05 were very low concentrations, biosorption wasdetermined at high values.

C. luteola TEM 05 was immobilized in carrageenan gels via entrap-ment and then used in the biosorption of heavy metals in batch reactors.

Figure 2. Single and partial competitive adsorption of Crþ6, Cdþ2 and Coþ2 oncarrageenan-chitosan (a) and C. luteola TEM 05 immobilized in carrageenan-chitosan gels (b). [^; Co-Mix, &; Cr-Mix, ~; Cd-Mix, �; Co, &; Cr, D; Cd].

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The competitive adsorbtion of Crþ6, Cdþ2, and Coþ2 from a mixture wasalso investigated. These experiments were performed at a constant pH(6.0) and at 25�C using solution containing 100mg L�1 from each metalion. The carrageenan and carrageenan-chitosan gels, with=withoutbiomass in metal solutions, were added. The results of biosorption studiesare given in Fig. 1 for carrageenan gels without biomass (a) and withbiomass (b) and also in Fig. 2 for carrageenan-chitosan gels withoutbiomass (a) and with biomass (b). As is seen from the figures, the compe-titive biosorption capacities of immobilized biomass for metal ions werelower than non-competitive conditions. According to these results, thereis approximately 3 times and 2 times decrease for partial competitive bio-sorption of Coþ2 and Cdþ2 on carrageenan-biomass, respectively. How-ever, non-competitive metal biosorption values are relatively close tocompetitive metal biosorption values for the carrageenan-chitosan gelswith biomass. In addition, the saturation levels for all metals were deter-mined approximately between 60–120min. The results agree well with theliterature.[1,20]

CONCLUSION

Biomass immobilization is an essential step for an industrial scale-upof biosorption. Unlike biomass in its native form, immobilizationprovides biosorbent particles with the adequate size density andmechanical strength reqired by continious systems. Natural polysac-charide gel matrices are widely used for this purpose and are a costeffective alternative to synthetic polymers. Carrageenan gels are verysuitable materials since cells can survive and can grow in the support,the diffusion coefficients are high, and the immobilization proceduresare easy to scale up. They are, however, soluble, biodegradable, andrelatively weak and, therefore, not suitable for treatment of waste-water. While the mixed solution of potassium chloride and chitosanwas used to gelate with carrageenan, the characteristics of the gelswere greatly improved. The carrageenan entrapped C. luteolaTEM 05 may be considered a good candidate for the biosorptionof metals. The findings are encouraging and should be applicableto industrial wastewater biosorption.

ACKNOWLEDGMENT

This work was supported by Ege University Research Fund Project 2004FEN 005.

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Received November 30, 2008Accepted December 26, 2008Manuscript 7586

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