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Colloids and Surfaces B: Biointerfaces 103 (2013) 381– 390

Contents lists available at SciVerse ScienceDirect

Colloids and Surfaces B: Biointerfaces

jou rn al h om epage: www.elsev ier .com/ locate /co lsur fb

omparative study of the kinetics and equilibrium of phenol biosorptionn immobilized white-rot fungus Phanerochaete chrysosporiumrom aqueous solution

iktor Farkasa,b, Attila Felingera,b, Alzbeta Hegedusovac, Imre Dékányd,e, Tímea Pernyeszia,b,∗

Analytical Chemistry and Geoanalytical Research Group, Szentágothai Research Center, University of Pécs, H-7624 Pécs, Ifjúság útja 20., HungaryDepartment of Analytical and Environmental Chemistry, Faculty of Science, University of Pécs, H-7624 Pécs, Ifjúság útja 6., HungaryDepartment of Vegetables Production, Slovak University of Agriculture in Nitra, SK-7624 Nitra, SlovakiaSupramolecular Nanostructured Materials Research Group of the Hungarian Academy of Sciences, University of Szeged, Aradi Vértanúk tere 1., H-6720 Szeged, HungaryDepartment of Medical Chemistry, Faculty of Medicine, University of Szeged, Aradi Vértanúk tere 1., H-6720 Szeged, Hungary

r t i c l e i n f o

rticle history:eceived 4 June 2012eceived in revised form 1 September 2012ccepted 6 September 2012vailable online 29 September 2012

a b s t r a c t

In this study the kinetics and equilibrium of phenol biosorption were studied from aqueous solution usingbatch technique at an initial pH of 5.5. The biosorption was studied on Ca–alginate beads, on non-livingmycelial pellets of Phanerochaete chrysosporium immobilized on Ca–alginate, and on free fungal biomass.Ph. chrysosporium was grown in a liquid medium containing mineral and vitamin materials with complexcomposition. The biosorption process followed pseudo second-order kinetics on all bioadsorbents. Thebioadsorption-equilibrium on blank Ca–alginate, free and immobilized fungal biomass can be described

eywords:iosorptionhenolh. chrysosporiumineticsquilibrium

by Langmuir, anti-Langmuir and Freundlich isotherm models using nonlinear least-squares estimation.© 2012 Elsevier B.V. All rights reserved.

onlinear least-squares estimation

. Introduction

Phenol is widely present in wastewaters discharged from indus-ries and it is considered as priority pollutant because of its highoxicity at low concentrations as well. Industrial sources of contam-nants such as oil refineries, coal gasification sites, petrochemicalnits, and so on, generate large quantities of phenols. In addi-ion, phenol derivatives are widely used as intermediates in theynthesis of plastics, colors, pesticides, insecticides, etc. [1]. Inrder to keep waters free from phenol compounds, different purifi-ation methods such as adsorption, chemical oxidation, solventxtraction, or reverse osmosis are used for removing phenols fromastewaters [2].

Biosorption, as an efficient, cost-effective and environmen-ally friendly technique – for heavy metals and various organic

ollutants – has emerged as a potential alternative to the con-entional techniques [3–6]. Biomass of some natural microbialpecies, including bacteria, fungi and algae, is capable of removing

∗ Corresponding author at: H-7624 Pécs, Ifjúság útja 6., Hungary.el.: +36 72 503600; fax: +36 72 501518.

E-mail address: ptimea@gamma.ttk.pte.hu (T. Pernyeszi).

927-7765/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.colsurfb.2012.09.029

the different organic pollutants by biosorption, biodegradation ormineralization [7,8].

Growing attention is being given to the potential health haz-ard presented by heavy metals to the environment. Uluozlu et al.used lichen biomass for the removal of different heavy metalsfrom aqueous solution. They studied the effect of different param-eters, such as pH, biomass dosage, contact time, and temperature.[9]. Biosorption of Cd (II) and Cr (III) from aqueous solution onHylocomium splendens biomass were investigated by Sari et al.They used different kinetic and isotherm models to evaluate theexperimental data. They found that the Langmuir model fitted theequilibrium data better than the Freundlich one. The use of theDubinin–Rhadushkevich model demonstrated that the process ision-exchange [10]. Lactofungus scrobiculatus biomass was used forheavy metal removal by Anayurt et al. The kinetic studies indicatedthat the process followed well pseudo second-order model. Therecovery of the metal ion was found as higher than 95% [11].

Phanerochaete chrysosporium is a well-known white-rot fungusand it has a strong ability to degrade xenobiotics [12,13]. Relatively

few studies have been carried out with Ph. chrysosporium in detox-ifying metal effluents and effluents containing phenol derivatives[6,7,14–19]. Most studies on bioadsorption were carried out withpowdered biomass and batch systems. The powdered biomass is

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82 V. Farkas et al. / Colloids and Surfac

ifficult to use in applications due to its disadvantages, the smallarticle size and low mechanical strength, which may cause diffi-ulty in the separation of biomass after biosorption and significantass loss after regeneration.The immobilization of native microorganisms on natural or syn-

hetic polymers improves their mechanical strength, rigidity, size,orosity characteristics, and resistance to environmental restrains.he selection of immobilization matrix is crucial in the applica-ion of immobilization biomass. The polymer matrix determineshe mechanical strength, rigidity, and porosity characteristics ofhe immobilized beads and their adsorption capacity. The physi-al entrapment of microorganisms inside a polymer matrix is onef the most widely used techniques or immobilization [20–27].odium alginate, chitin, chitosan, and cellulose derivatives asatural polymers have been used as the matrices for immobiliza-ion [20–27]. Immobilized biomass exhibits a greater potential inxed/fluidized bed reactors because of minimal clogging underontinuous flowing conditions, convenience for regeneration, reusef biomass and easy solid–liquid separation [3,26,27].

For the immobilization of Ph. chrysosporium, little informa-ion exists in the literature. Yu and Wu [25] used alginate, pectinnd polyvinyl alcohol (PVA) to immobilize Ph. chrysosporiumells for 2,4-dichlorophenol bioadsorption. The immobilization ofh. chrysosporium onto pectin was less efficient than that ontother matrices because of its poor mechanical strength and lowdsorption efficiency. Ca-alginate immobilized fungal beads withiocompatibility exhibited good mechanical strength and adsorp-ion efficiency over 60%. Among the different biomass dosage ina–alginate immobilized fungal beads 1.25% (w/v) was the opti-um [20,25].The continuous-flow adsorption of 2,4-dichlorophenol (2,4-

CP) from aqueous solution on immobilized Ph. chrysosporiumiomass in a fixed-bed column was also studied [26]. Theyound that the breakthrough time decreased with increasing flowate, increasing influent concentration, and decreasing bed depth.he data also indicated that the equilibrium uptake of 2,4-DCPncreased with decreasing flow rate and increasing influent con-entration of 2,4-DCP.

Mathialagan and Viraraghavan studied the biosorption of pen-achlorophenol (PCP) on treated Aspergillus niger biomass. Theyound that the biosorption was pH dependent. The removal of PCPecreased with the increase of pH for all types of biomass. They alsovaluated the results with different kinetic and isotherm models8,28].

Ca–alginate and loofa sponge as a support for lead (II), copperII), and zinc (II) biosorption on immobilized Ph. chrysosporium waslso studied [20,21,23]. Iqbal and Saeed reported the possibility ofmmobilizing Ph. chrysosporium on a biostructural matrix of loofaponge as a dye biosorbent system for the removal of RBBR (Rema-ol Brilliant Blue R), a reactive dye, from aqueous solution. Theeactive dye uptake from aqueous solution was found to be influ-nced by solution pH, temperature and initial dye concentration23].

The fibrous network of Papaya wood as a special immobiliza-ion matrix was used to immobilize Ph. chrysosporium by Iqbal andaeed [22]. They used this matrix for removal of zinc (II) from aque-us solution. They obtained that the immobilized fungal biosorbentemoved zinc (II) rapidly and efficiently with a maximum metalemoval capacity of 66.17 mg g−1 at equilibrium, 41.93% higherhan the amount of zinc (II) removed by free biomass. The sorp-ion data agreed well with the second-order kinetic model, thequilibrium data fitted very well to the Langmuir model.

The main objective of the present study was to immobilizenactive Ph. chrysosporium fungal biomass in Ca–alginate poly-

er matrix and evaluate the phenol bioadsorption kinetics andquilibrium on blank Ca–alginate, immobilized Ph. chrysosporium

iointerfaces 103 (2013) 381– 390

fungal biomass with comparison of the adsorption data on free Ph.chrysosporium biomass, The effect of pH, adsorption time, initialphenol concentration on bioadsorption capacity was studied in abatch system.

2. Experimental

2.1. Chemicals

All chemicals used were of analytical grade. Na–alginate waspurchased from Sigma Aldrich Ltd., Germany. Phenol (>99% purity)was purchased from Sigma–Aldrich Ltd., Germany and was usedwithout further purification. Stock solutions were prepared by dis-solving 0.2 g of phenol in 1.0 L of distilled water. The test solutionscontaining phenol were prepared by diluting 200 mg L−1 of stocksolution of phenol to the desired concentrations. The pH value of thebiosorption systems (2.0–7.0) was adjusted to the required valueusing 0.1 M NaOH or 0.1 M HCl (Merck AG., Germany) solutions. Allsolutions were stored in the dark at 4 ◦C prior to use.

2.2. Cultivation of fungal biomass

Ph. chrysosporium (strain SzMC 1726) obtained from the Depart-ment of Environmental Microbiology, Faculty of Science, Universityof Pécs (Hungary) was used in this study. It was cultivated as previ-ously described by Kirk et al. [29]. After 5 days incubation at 35 ◦Con a shaker (app. 180 rpm), the mycelial pellets were removed fromthe medium through filtration and inactivated in a pressure cookerat high temperature (120 ◦C) for 20 min. Then the mycelia pelletswere washed several times with deionized water. These mycelialpellets were immobilized in the next step.

2.3. Immobilization of Phanerochaete chrysosporium inCa–alginate beads

For immobilization of fungal biomass, the optimum concentra-tion of Ph. chrysosporium biomass and Na–alginate solution wasdetermined by Wu and Yu [25]. The optimized concentration ofbiomass in Ca–alginate was 1.25% and the optimized concentra-tion of Na–alginate solution was 2%. For the immobilization ofbiomass, these concentration values were used. Fungal suspen-sion was dropped into a 0.2 M CaCl2 solution, and the drops ofalginate–biomass mixture were later gelled into beads with a meandiameter of 3–4 mm. The Ca-alginate immobilized Ph. chrysospo-rium beads were stored in the CaCl2 solution at fridge temperaturearound 5 ◦C for 4 h to cure. Then the beads were rinsed severaltimes with deionized water and stored in fridge prior to use. Forblank Ca–alginate beads, similar procedures were used but withoutfungal biomass.

2.4. Biosorption study

The biosorption of phenol onto the blank Ca–alginate beads, freeand immobilized fungal beads was investigated in batch biosorp-tion systems.

For comparison, 0.075 g of free biomass (dry weight), immo-bilized fungal beads containing 0.075 g biomass (dry weight) andblank beads (dry weight) were respectively mixed with 250 mLphenol solution at the concentrations of 25 and 50 mg L−1. Thesample holder was agitated on a shaker at 400 rpm at room tem-perature. Samples were taken at given time intervals, and then

centrifuged at 10,000 rpm for 5 min. The supernatant was used foranalysis.

For equilibrium studies, the blank beads, the free and immobi-lized fungal biomass were respectively put into phenol solutions

V. Farkas et al. / Colloids and Surfaces B: Biointerfaces 103 (2013) 381– 390 383

Fig. 1. Stereo microscopic view (a) of Ca–alginate bead, free biomass, and immobilized biomass. Scanning electron microscopic image of (b) the Ca–alginate bead (at 70×m and (da

wt5Te

q

wpct

2

est1cU2

timfluc1

agnification), (c) of the surface of the Ca–alginate bead (at 1000× magnification)

re about 5 mm.

ith initial concentrations from 10 to 100 mg L−1. The concen-ration of biosorbent was 0.3 g L−1, the suspension volume was0 mL. The experiments were carried out at 22.5 ◦C temperature.he adsorbed amount of phenol was calculated with the followingquation:

= (C0 − Ce)Vm

(1)

here q is the adsorbed amount of phenol (mg g−1); C0 is the initialhenol concentration (mg L−1); Ce is the equilibrium phenol con-entration (mg L−1); V is the volume of the solution (L); and m ishe weight of the biosorbent (g).

.5. Analysis

HPLC (Agilent Technologies, USA) was used to determine thequilibrium concentration of phenol in the supernatant. The HPLCystem contained a degasser (Agilent 1100 Series), a system con-roller (Agilent 1200 Series), an autosampler and injector (Agilent200 Series). The Chemstation software was used to evaluate thehromatographic data. The experiments were performed with aV/vis photo diode array detector at the wavelengths of 217 and70 nm.

A Waters Symmetry C18 column (4.6 mm × 150 mm; 5 �m par-icle size) was used. All the measurement were carried out insocratic mode using the mobile phase composition 60:40% (v/v),

ethanol:water). The separation conditions were the following:

ow rate 1 mL min−1, column temperature 50 ◦C, and injection vol-me 20 �L for both the standard and samples as well. Calibrationurve of the standard was made from stock solution in the range of0–100 mg L−1.

) of the hyphal structure of free Ph. chrysosporium biomass. Diameters of the beads

2.6. Morphological study with scanning electron microscope(SEM)

SEM studies were conducted in the Central Electron MicroscopeLaboratory, Faculty of Medicine, University of Pécs. A Jeol JSM-6300(Jeol Ltd., Japan) scanning electron microscope was used in thisstudy.

Samples were lyophilized as a drying procedure. No further fixa-tion procedures were done during the sample preparation protocol.Before the samples had been covered with gold, dehydrated gran-ules were fixed on a microscope slide (Fig. 1).

3. Results and discussion

3.1. The effect of initial pH on phenol bioadsorption onCa–alginate, immobilized and free Ph. chrysosporium biomass

Earlier studies on biosorption have shown that pH is an impor-tant parameter affecting the biosorption process [30]. The effectof the pH of the initial solution on the phenol uptake capacity ofthe adsorbents (Ca–alginate blank beads, Ca–alginate immobilizedPh. chrysosporium biomass, and free Ph. chrysosporium biomass)was studied in the pH range 2.0–7.0 at a suspension concentrationof 0.3 g L−1. One can see in Fig. 2a–c that the biosorption of phe-nol first increases from pH 2.0 and then declines with the furtherincrease of pH. For Ca–alginate beads, the maximum equilibriumphenol uptake was found at 1.21 and 2.22 mg g−1, respectively,for 25 and 50 mg L−1 at pH 6 (Fig. 2a) For alginate immobilizedfungal biomass, the maximum equilibrium uptake was found at

2.06 and 3.93 mg g−1, respectively, for 25 and 50 mg L−1 phe-nol concentrations at pH 6 (Fig. 2b). For free Ph. chrysosporiumbiomass, the maximum equilibrium uptake was found at 2.82and 5.25 mg g−1, respectively, for phenol concentrations of 25 and

384 V. Farkas et al. / Colloids and Surfaces B: B

Fig. 2. The effect of pH on phenol biosorption process by (a) Ca–alginate, (b) immobi-lized Ph. chrysosporium biomass and (c) free biomass at initial phenol concentrationsoE

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f 25 and 50 mg L−1 in aqueous suspension. The biomass concentration is 0.3 g L−1.rror bar represents standard deviation (SD); n = 3.

0 mg L−1 (Fig. 2c). During the adsorption process the adjusted pHalue of the suspensions did not significantly varied.

Similar trends were observed for two initial concentrationsn the case of Ca–alginate, immobilized and free biomass.

ith increasing initial concentration, the phenol uptake capacityncreased in all cases. In the case of free biomass and immobilizediomass, in the natural state of pH 5.0–6.0, the adsorption of phenolas at the maximum level. In the case of Ca–alginate immobilized

iomass, the adsorption was at maximum at pH 6.0. The bioadsorp-

ion was slightly reduced in alkaline and acidic medium. The effectf pH on phenol bioadsorption was not significant in the pH range of.0–8.0, and the uptake of phenol in that pH range was larger thant other pH values. The pH of the solution influences both the cell

iointerfaces 103 (2013) 381– 390

surface binding sites and chemistry in water. Earlier studies foundthat the adsorbed amount correlated with the dissociation con-stant of phenol derivatives [7,15,19]. The ionic fraction of phenolateion increases with increasing pH and the surface charge of fungalbiomass is predominantly negative at pH 3.0–10.0 [7,15,19,31]. Itwas also reported that the electrostatic forces between the chargedfungal surface and phenol played an important role in the bioad-sorption process.

In our earlier study, the influence of pH on phenol bioadsorp-tion was also investigated on free dead Ph. chrysosporium biomass,but the cultural medium had a different composition with a simpleconstitution. The maximal uptake capacity was obtained at pH 5.5.In the present study, the cultural medium for fungal biomass has acomplex composition [32]. Wu and Yu [25,33] found that the max-imum uptake capacity for 2,4-dichlorophenol was at pH 5.0–6.0 forboth free and immobilized fungal biomass.

3.2. Bioadsorption kinetics of phenol on Ca-alginate beads,immobilized Ph. chrysosporium biomass, and free biomass fromaqueous suspension

The biosorption time of phenol onto Ca–alginate beads (2%),alginate immobilized Ph. chrysosporium biomass, and free Ph.chrysosporium biomass was evaluated with a solution contain-ing 50 mg L−1 of phenol at pH 5.5 in natural state without pHadjustment. The biosorbent concentration was 0.3 g L−1, the con-centration of Ph. chrysosporium biomass was 1.25% in the alginatebeads. The initial phenol concentration was 50 mg L−1. The changeof phenol concentration in the supernatant is presented in Fig. 3a.In Fig. 3b, the adsorbed amounts of phenol, and in Fig. 3c theadsorption efficiency (in percent) is presented against the sorp-tion time. Fig. 3a–c demonstrate that the adsorption rate wasinitially high in the very first minutes, and the saturation wasreached after sixty minutes for all the adsorbents studied. Beyondthat time, the amount of adsorbed phenol did not increase signif-icantly with sorption time. At the bioadsorption equilibrium withinitial phenol concentration of 50 mg L−1, the adsorption capacitywas 2.78 mg g−1 on Ca–alginate beads, 3.33 mg g−1 on immobi-lized biomass and 6.73 mg g−1 on free biomass. Fig. 3c also showsthat approximately 13.45% of phenol equilibrium uptake could bereached within forty minutes on free fungal biomass, and that thecorresponding phenol equilibrium uptake was 6.66% and 4.85% forimmobilized fungal biomass and blank Ca–alginate beads, respec-tively. This suggests that the adsorption rate of phenol onto theimmobilized fungal beads was slower than that onto the free fun-gal biomass in the initial biosorption period. In our study, the lowadsorption efficiency can be explained with the low biosorbentdosage. Wu and Yu [25] reported that approximately 78.03% of2,4-DCP equilibrium uptake could be achieved within 30 min onthe free fungal biomass, and that the corresponding 2,4-DCP equi-librium uptake was 68.04% and 71.24% for the immobilized fungalbiomass and Ca–alginate beads, respectively, in the case of biomassdosage of 5 g L−1 and initial concentration of 40 mg L−1. We haveto consider that the adsorption increases with decreasing watersolubility of the molecule and with increasing octanol–water parti-tioning coefficient. The free biomass had its binding sites exposed tophenol, whereas the entrapped biomass was retained in the interiorof the immobilized beads [20,25]. The time of 60 min was consid-ered to be sufficient for phenol biosorption to reach equilibrium forall the adsorbents studied.

3.3. Kinetic modeling

There are several kinetic models regarding the adsorption ofheavy metals, dyes and chlorophenols [3,7,31,32,34]. To evalu-ate the bioadsorption kinetics of phenol, two kinetic models were

V. Farkas et al. / Colloids and Surfaces B: Biointerfaces 103 (2013) 381– 390 385

Fig. 3. Time courses of phenol biosorption by Ca–alginate, immobilized Ph.chrysosporium biomass beads and free biomass at pH 5.5, T = 22.5 ◦C. (a) The phenolconcentrations are presented against the sorption time. (b) The adsorbed phenolamounts are presented against the sorption time. (c) The adsorption efficiency val-ues are presented against the sorption time. The initial phenol concentration is5b

uCc

3

e

Fig. 4. (a) Linearized pseudo first-order kinetic model for phenol sorption byCa–alginate beads, immobilized biomass in Ca–alginate and free Ph. chrysosporiumbiomass. (b) Linearized pseudo second-order kinetic model for phenol sorption byCa–alginate beads, immobilized biomass in Ca–alginate and free Ph. chrysosporium

The pseudo second-order kinetic rate equation can be written

0 mg L−1 and the biomass dosage is 0.3 g L−1. The biomass concentration in alginateeads is 1.25%. Error bar represents SD; n = 3.

sed to fit the experimental data obtained on Ca–alginate beads,a–alginate immobilized Ph. chrysosporium biomass, and free Ph.hrysosporium biomass.

.3.1. Pseudo first order-Lagergren modelThe pseudo first-order kinetic model (Lagergren model) is gen-

rally expressed as follows:

dq

dt= k1,ad(qeq − q) (2)

biomass. The initial phenol concentration is 50 mg L−1 and the biomass dosage is0.3 g L−1. The biomass concentration in alginate beads is 1.25%. Error bar representsSD; n = 3.

where k1,ad is the adsorption rate constant of first order bioad-sorption (min−1), q is adsorbed amount (mg g−1), qeq is adsorptioncapacity (mg g−1) at equilibrium.

Integration and linearization of Eq. (2) give

log(qeq − q) = log qeq − k1,adt

2.303(3)

The plots of log(qeq − q) vs. sorption time are shown in Fig. 4a.The linear relationships were observed only for the initial 30 min ofsorption and the experimental data considerably deviated from thetheoretical ones after this initial period. The sorption rate constantsk1,ad and the theoretical values of qeq calculated from the slope andintercept of the linear plots are summarized in Table 1, with thecorresponding correlation coefficients.

The sorption rate constant k1,ad varied in the range of 1.15 × 10−2

to 6.91 × 10−2 min−1. The first-order rate constants k1,ad are5.13 × 10−2 min−1 for Ca–alginate beads, 1.15 × 10−1 min−1 for theimmobilized biomass and 6.91 × 10−2 min−1 for the free biomass.

The theoretical values of sorption capacity qeq are lower than theexperimental values. The calculated adsorption capacities qeq,cal are2.20 mg g−1 for Ca–alginate beads, 1.81 mg g−1 for the immobilizedbiomass and 2.69 mg g−1 for the free biomass.

3.3.2. Pseudo second-order kinetic model

as follows:

dq

dt= k2,ad(qeq − q)2 (4)

386 V. Farkas et al. / Colloids and Surfaces B: Biointerfaces 103 (2013) 381– 390

Table 1The pseudo first- and second-order rate constants and the calculated equilibrium adsorption capacities on free Ph. chrysosporium biomass, Ca–alginate immobilized biomass,and Ca–alginate beads at pH 5.5. Ca–alginate concentration: 2%, the biomass concentration: 1.25%. T = 22.5 ◦C, biomass concentration: 0.3 g L−1.

Biosorbent k1,ad (min−1) qeq,cal (mg g−1) R2 k2,ad (min−1) qeq,cal (mg g−1) R2 qeq,exp (mg g−1)

−1

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Free biomass 6.91 × 10 2.69 0.946

Alginate + biomass 1.15 × 10−1 1.81 0.963

Alginate 5.13 × 10−1 2.20 0.852

where k2,ad is the rate constant of second order bioadsorp-ion (g mg−1 min−1), q is the adsorbed amount (mg g−1), qeq is thedsorption capacity (mg g−1) in the equilibrium.

The integrated and linearized form of Eq. (4) is

t

q= 1

k2,adq2eq

+ t

qeq(5)

For the utilization of this model, the experimental value of qeq isot necessary to be estimated a priori. Straight lines were obtainedy plotting t/q against t for Ca–alginate beads, for immobilizediomass in Ca–alginate, and free biomass (Fig. 4b). The second-rder rate constants, k2,ad and qeq, are summarized in Table 1nd were determined from the slope and intercept of the plotsFig. 4a and b). The sorption rate constant k2,ad varies in the rangef 5.47 × 10−1 to 4.27 g mg−1 min−1.

The second-order rate constants, k2,ad, are.47 × 10−1 g mg−1 min−1 for Ca–alginate, 1.62 g mg−1 min−1

or the immobilized biomass and 4.27 g mg−1 min−1 for the freeiomass. The largest second-order rate value (4.27 g mg−1min−1)as obtained for the free biomass. The Ph. chrysospo-

ium biomass immobilized in Ca–alginate exhibits a higherate constant (1.62 g mg−1 min−1) than the alginate beads5.47 × 10−1 g mg−1 min−1).

The theoretical adsorption capacities qeq,cal are 2.94 mg g−1 fora–alginate beads, 3.41 mg g−1 for the immobilized biomass, and.85 mg g−1 for the free biomass. The calculated adsorption capac-

ties agreed well with the experimental data. The correlationoefficients for the pseudo first-order kinetics model were lowerhan for the pseudo second-order one.

From the comparison of the two kinetic models, we can concludehat the biosorption process of phenol onto the surface of Ca-lginate, immobilized Ph. chrysosporium biomass, and free biomassollows the pseudo second-order kinetics. Dursun and Kalayci [35]tudied the phenol adsorption on chitin from aqueous solution at

biosorbent dosage of 1 g L−1 (29). They found that the adsorp-ion process followed pseudo second-order kinetic model, andhe adsorption rate constant varied in the range of 3.9 × 10−3 to.5 10−3 g mg−1 min−1 at 20, 30 and 40 ◦C.

.4. Bioadsorption isotherms of phenol on Ca–alginate beads,mmobilized Ph. chrysosporium biomass, and free biomass fromqueous suspension

The biosorption isotherms of phenol onto Ca–alginate beads2%), immobilized Ph. chrysosporium biomass in Ca–alginate, andree Ph. chrysosporium biomass were determined by varying theirnitial concentrations in the range of 10–100 mg L−1 with a con-tant adsorbent dosage of 0.3 g L−1 at pH 5.5 in natural state withoutH adjustment. The Ph. chrysosporium biomass concentration was.25% in the Ca–alginate beads and the Ca–alginate concentrationas 2%. In Fig. 5a and b the change of adsorbed amount of phenol

n all the adsorbents are presented against equilibrium concen-rations. The free biomass has a higher adsorption capacity than

he immobilized biomass in Ca–alginate and the blank biomass.he experimental maximum adsorbed amount was 3.27 mg g−1

or blank alginate beads, 7.81 mg g−1 for immobilized biomass ina–alginate and 13.50 mg g−1 for the free biomass at the initial

4.27 6.85 0.996 6.731.62 3.41 0.999 3.335.47 × 10−1 2.94 0.996 2.78

phenol concentration of 100 mg L−1. The experimental results arecompared with other studies such as bioadsorbent and adsorp-tion capacity in Table 2. Biosorption of 2,4-dichlorophenol by freePh. chrysosporium biomass, immobilized biomass in alginate (2%)and alginate beads from aqueous suspensions were studied by Wuand Yu [25]. The theoretical maximum adsorption capacities deter-mined from the Langmuir model were 1.63, 4.55, and 7.15 mg g−1

for the blank Ca–alginate, immobilized, and free fungal biomass,respectively.

3.5. Modeling the equilibrium of bioadsorption

Among the zillions of two- and three-parameter isothermequations, two simple models are frequently used to describebiosorption processes [3]. To evaluate the biosorption isothermsof phenol, we used the Freundlich, Langmuir, and anti-Langmuir[36] models to fit the experimental equilibrium data determinedon blank Ca–alginate beads, immobilized biomass in Ca–alginatebeads, and free Ph. chrysosporium biomass. In this study, the non-linear least-squares estimation and the linearized presentation ofisotherm equations were used for the evaluation of bioadsorptionequilibrium. In the research field of biosorption, the linearized pre-sentation of isotherm equations has been frequently used for thedetermination of bioadsorption constants despite of its hazards[3,37]. We present a comparison of the equilibrium constants cal-culated from the linearized and original, nonlinear forms of theisotherm equations.

3.5.1. Freundlich isotherm modelThe well-known Freundlich model is expressed as follows:

qeq = KF C1/ne (6)

where qeq is the adsorbed amount in the equilibrium (mg g−1); KF

is the Freundlich constant (mg g−1); Ce equilibrium concentrationof phenol (mg L−1)

Linearization of Eq. (6) gives

log qe = log KF + 1n

log Ce (7)

The plots of logqe against logCe are shown in Fig. 6a. We havecompared the Freundlich constants calculated from the linearizedpresentation of the isotherms with the nonlinear least-squaresestimates (Fig. 5a and a, Table 3) [37]. The values of the Freund-lich constant KF and the exponent n calculated by Eqs. (6) and(7), respectively, and the corresponding correlation coefficientsare summarized in Table 3. From the linearized presentation theFreundlich constant, KF varied in the range of 0.023–0.203 mg g−1.The Freundlich constants KF are 2.03 × 10−1 mg g−1 for freebiomass, 4.73 × 10−2 mg g−1 for the immobilized biomass, and2.30 × 10−2 mg g−1 for blank Ca–alginate beads. The order of mag-nitude of KF was: free fungal biomass > immobilized fungi inCa–alginate beads > blank Ca–alginate beads. This order indicatesa higher adsorption capacity of the free and immobilized fungal

beads over the blank Ca–alginate beads. For free biomass, the n val-ues were greater than unity, indicating a favorable adsorption. Forimmobilized fungal biomass and blank Ca–alginate, the n values areclose to unity. The values of exponent n are 1.16 for free biomass,

V. Farkas et al. / Colloids and Surfaces B: Biointerfaces 103 (2013) 381– 390 387

Fig. 5. (a) Fitted phenol bioadsorption isotherms from nonlinear least-square estimation using Freundlich equation (b) data from nonlinear least-square estimation usingL beadsL nted ab

0b

eb3vibI–i

TC

angmuir equation by Ca–alginate beads, immobilized Ph. chrysosporium biomass

angmuir isotherms using calculated data. The adsorbed phenol amounts are presear represents SD; n = 3.

.93 for the immobilized biomass and 0.91 for blank Ca–alginateeads.

The estimated values of constant KF using nonlin-ar least-squares fitting are 2.92 × 10−2 mg g−1 for freeiomass, 8.73 × 10−3 mg g−1 for the immobilized biomass, and.70 × 10−2 mg g−1 for blank Ca–alginate beads. The estimatedalues of exponent n are 0.75 for free biomass, 0.67 for themmobilized biomass and 1.01 for blank Ca–alginate beads. For

oth fitting methods the correlation coefficients are rather high.

n Fig. 5c, the experimentally determined and the fitted isotherms using the Freundlich constants calculated from the linearized

sotherm equation – are presented. Nevertheless, we can conclude

able 2omparison of biosorption capacity on various biosorbents for phenol and its derivatives

Type of adsorbents Pollutant Equilibriumtime

Pseudomonas putida + activated sludge Phenol 48 h

Caulerpa scalpelliformis Phenol 6 h

Funalia trogii Phenol, 2-chlorophenol 6 h

Pleurotus sajor caju Chlorophenols 4 h

Activated carbon Phenol 30 days

Bacillus subtilis Phenol 30 min

Chitosan–calcium beads Phenol, o-chlorophenol 4 h

Aspergillus niger Phenol 24 h

Dried sewage sludge Phenol 24 h

Phanerochaete chrysosporium Phenol 24 h

Ca–alginate beads (2%) Phenol 24 h

Immobilized Phanerochaete chrysosporium Phenol 24 h

Phanerochaete chrysosporium 2,4-DCP 6 h

Ca–alginate beads (2%) 2,4-DCP 6 h

Immobilized Phanerochaete chrysosporium 2,4-DCP 6 h

and free biomass at pH 5.5, T = 22.5 ◦C. (c) Presentation of the Freundlich and (d)gainst the equilibrium concentration of phenol after the biosorption process. Error

that the nonlinear least-squares estimation using the Freundlichmodel gives better fit and more authentic results (Fig. 5a and c)than the linearization.

3.5.2. Langmuir and anti-Langmuir isotherm modelThe Langmuir model is valid for monolayer adsorption onto a

surface containing limited number of identical sites [3]. The Lang-

muir isotherm model is written in the following form:

qeq = qmaxKLCe

1 + KLCe(8)

.

Pollutant/adsorbentconcentration (g L−1)

pH Adsorption capacity (mg g−1) References

(0.1–0.5)/– 7.0 80% phenol removal [38]0.1/6 6.0 20.1 [39]0.2/(0.25–2.0) 8.0 132.6, 289.1 [40]0.5/0.2 6.0 0.95–1.89 mmol/g [41]– 6.0 303 [42]0.015/10 0.2 0.06 [43]0.3/1 7.0 116.3, 64.9 [44]0.001/2.7 5.1 0.33 [31]0.1/5 6.5 17.3 [45]0.1/0.3 6.0 13.5 Present study0.1/0.3 6.0 3.27 Present study0.1/0.3 6.0 7.81 Present study0.04/5 5.0 3.22 [25]0.04/5 5.0 0.93 [25]0.04/5 5.0 2.13 [25]

388 V. Farkas et al. / Colloids and Surfaces B: Biointerfaces 103 (2013) 381– 390

Table 3The Freundlich and Langmuir constants calculated from the nonlinear least-squares estimation and linearized presentation for the adsorption of phenol onto the blankCa–alginate beads, immobilized fungal biomass in Ca–alginate and free biomass at pH 5.5. Ca–alginate concentration: 2%, the biomass concentration: 1.25%. T = 22.5 ◦C,biomass concentration: 0.3 g L−1.

Phenol biosorption From nonlinear least-squares estimation From linearized presentation

Freundlich isotherm model

KF (mg g−1) n R2 KF (mg g−1) n R2 qmax,exp (mg g−1)

Free biomass 2.92 × 10−2 0.75 0.963 2.03 × 10−1 1.16 0.921 13.50Alginate + biomass 8.73 × 10−3 0.67 0.982 4.73 × 10−2 0.93 0.948 7.81Alginate 3.69 × 10−2 1.01 0.985 2.30 × 10−2 0.91 0.991 3.27

Anti-Langmuir and Langmuir isotherm model

KL (L mg−1) qmax,cal (mg g−1) R2 KL (L mg−1) qmax,cal (mg g−1) R2 qmax,exp (mg g−1)

−3 −3

w(ic

FiLicT

Free biomass 4.35 × 10 – 0.981

Alginate + biomass 4.70 × 10−3 – 0.980

Alginate 6.47 × 10−4 57.16 0.964

here qeq is the adsorbed amount of phenol in the equilibriummg g−1); KL is the Langmuir constant (L mg−1); qmax is the max-mal adsorbed amount of phenol (mg g−1); Ce is the equilibriumoncentration of phenol (mg L−1)

The linearization of Eq. (8) gives

1qeq

=(

1KLqmax

)1Ce

+ 1qmax

(9)

ig. 6. (a) Linearized Freundlich isotherm for phenol sorption by Ca–alginate beads,mmobilized biomass in Ca–alginate beads and free Ph. chrysosporium biomass. (b)inearized Langmuir isotherm model for phenol biosorption by Ca–alginate beads,mmobilized biomass in Ca–alginate beads and free biomass. The initial phenol con-entration varied between 10 and 100 mg L−1 and the biomass dosage is 0.3 g L−1.he biomass concentration in alginate beads is 1.25%. Error bar represents SD; n = 3.

2.86 × 10 45.04 0.938 13.504.18 × 10−3 16.92 0.959 7.811.74 × 10−1 6.45 0.993 3.27

The so called anti-Langmuir model describes a concave upwardisotherm [36]. Its equation is:

q = qmaxCe

1 − KLCeKL > 0 (10)

The linearized plots of the Langmuir equation describing thebiosorption of phenol on blank Ca–alginate, immobilized fun-gal biomass in Ca–alginate, and free biomass are illustrated inFig. 6b. The values of the Langmuir constants were calculatedusing the least-squares fitting along with correlation coefficients(R2) and their estimated values from the nonlinear least-squaresestimation are summarized in Table 3 (Figs. 5b and 6b). The the-oretical maximum adsorption capacity defined the total capacityof the bioadsorbent for phenol. From the linearized presentation,the maximum adsorption capacities qmax were 6.45, 16.92 and45.04 mg g−1 for the blank Ca–alginate, immobilized, and free fun-gal biomass, respectively. The Langmuir constant KL is an indicatorof the stability of the combination between adsorbate and adsor-bent surface and a constant related to the free energy or netenthalpy of adsorption [20].

From the nonlinear least-squares estimation, the KL

values obtained using the anti-Langmuir equation were4.35 × 10−3 L mg−1 for free biomass, 4.70 × 10−3 L mg−1 forimmobilized biomass, respectively, which suggest an anti-Langmuir behavior in these cases. The equilibrium constantwas 6.47 × 10−4 L mg−1 for blank Ca–alginate beads. The maxi-mum adsorption capacity qmax was 57.16 mg g−1 for the blankCa–alginate in this method. Rather good correlation coefficientswere found (Table 3), implying that both methods could beused to describe the adsorption-equilibrium of phenol onto thebioadsorbents.

Nevertheless, from the comparison of the linearized and nonlin-ear least-squares estimation we can conclude that rather differentresults are obtained. In Fig. 5d, the experimentally determined andthe fitted isotherms – using the Langmuir constants calculated fromlinearized isotherm equation are presented. It can be seen that thecalculated isotherms for all biosorbents have significantly differentcurve shapes.

Due to the linearization step, the weights of the errors areentirely different, and fitting the linearized data gives false results.

The anti-Langmuir model is concave upward and so are theplots of the experimental data of phenol adsorption on free andimmobilized fungal biomass. Therefore, the anti-Langmuir modelis expected to fit and one cannot expect a good fit with the convex

upward Langmuir model. Still, the linearized data fitting proceduresuggests Langmuir-type behavior. Due to the hazards of equationlinearization, the nonlinear least-squares estimation can be sug-gested for modeling bioadsorption-equilibrium [37]. Besides, one

es B: B

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[

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V. Farkas et al. / Colloids and Surfac

hould always plot the fitted isotherm and the experimental dataq vs. Ce) for a visual check of the quality of fitting. Fig. 5d demon-trates that the results of the fitting of the linearized data may nott all represent the experimental ones.

. Conclusions

The comparison of adsorption capacity of Ca–alginate beads,mmobilized Ph. chrysosporium in Ca–alginate, and free dead Ph.hrysosporium for phenol was presented in this study. The max-mum adsorbed amounts of phenol were obtained at pH valuesf 5.0–6.0 for all tested bioadsorbents. The bioadsorption capacityecreased in the order of free dead biomass, immobilized biomass

n Ca–alginate beads, and Ca–alginate beads. However, the immobi-ization of biomass gives high mechanical strength, so there coulde an opportunity to use it in a possible fixed-bed reactor underow condition. Bioadsorption kinetics was evaluated using pseudorst- and second-order kinetic model. The bioadsorption of phenol

ncreased with increasing sorption time, the bioadsorption equilib-ium was reached within sixty minutes. The biosorption of phenoln free Ph. chrysosporium, immobilized fungal biomass and alginateeads followed pseudo second-order kinetics. Biosorption equilib-ium was evaluated by Freundlich, Langmuir, and anti-Langmuirsotherm model using linearized presentation and nonlinear least-quares estimation. The bioadsorption process of phenol follows annti-Langmuir behavior for free fungal biomass and immobilizedungal biomass. Due to the hazards of linearization the nonlinearstimation can be suggested for the evaluation of bioadsorptionquilibrium.

cknowledgements

Tímea Pernyeszi and Alzbeta Hegedusova gratefully acknowl-dge support for this research from the Hungarian-Slovakianntergovernmental and Cooperation Programme between Uni-ersity of Pécs and Constantine the Philosopher University for009–2012 (TéT 08-SK-2009-0013, SK-18/2008). Tímea Pernyeszi,ttila Felinger and Viktor Farkas acknowledge support for

his research from TAMOP-4.2.2./B-10/10-2010-0029 (Hungarianesearch grant).

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