Enzyme and Microbial Technology 39 (2006) 996–1001

Entrapment of fungal hyphae in structural fibrous network of papaya woodto produce a unique biosorbent for the removal of heavy metals

M. Iqbal ∗, A. SaeedEnvironmental Biotechnology Group, Biotechnology and Food Research Centre, PCSIR Laboratories Complex, Ferozepur Road, Lahore-54600, Pakistan

Received 12 August 2005; received in revised form 30 January 2006; accepted 7 February 2006

Abstract

A unique biosorbent was developed by entrapping fungal hyphae in structural fibrous network of papaya wood (SFNPW) and successfully usedfor the removal of Zn(II) from aqueous solution. The SFNPW-immobilized fungal biosorbent removed Zn(II) rapidly and efficiently with maximummetal removal capacity of 66.17 mg/g dry biomass at equilibrium, 41.93% higher than the amount of Zn(II) removed by free biomass (46.62 mg/g)under the identical conditions. Equilibrium was established in 1 h and biosorption was well defined by Langmuir isotherm model. SFNPW-immobilized fungal biosorbent was regenerated by washing with 50 mM HCl, with upto 99% recovery of the sorbed metal ions and the regeneratedsystem was reused in five adsorption–desorption cycles without any significant loss in its biosorption capacity. The efficient metal removing abilityof©

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f this unique biosorbent system, low cost of SFNPW and simplicity of the immobilization technique used to produce the SFNPW-immobilizedungal biosorbent system indicate the potential application of this biosorbent for the treatment of wastewaters containing heavy metals.

2006 Elsevier Inc. All rights reserved.

eywords: Biosorption; Immobilization; Papaya wood; Phanerochaete chrysosporium; Carica papaya; Zinc(II)

. Introduction

Biosorption by microorganisms such as algae, fungi, bacte-ia and yeasts has been suggested as an alternative procedureor the remediation of wastewaters contaminated with heavyetals [1]. Commercial application of these microorganisms asetal adsorbents (biosorbents) is, however, not feasible due to

roblems associated with their physical characteristics [2]. Lowensity and mechanical strength of the biomass can cause dif-culties in the separation of its dispersed solid-phase from the

iquid-phase effluents, which in turn contribute to limitationsn the development of cost-effective process design. A furtherroblem is associated with fragmentation of the hyphal biomassnd cell mass sedimentation causing flow restrictions in theontinuous-flow contact vessels. This has led to an interest inhe use of entrapped biomass as immobilized systems.

Several immobilization media, such as alginates, car-ageenans and polyacrylamide gels have been used for theurpose [3]. Due to their closed embedding structures, the immo-

∗ Corresponding author at: Biotechnology and Food Research Centre, PCSIR

bilization matrices based on these polymeric gels, however,result in reduced metal sorbate-biomass sorbent contact andrestricted diffusion [4]. Their use is further limited by theirinsufficient mechnical strength and the lack of open spaces toaccommodate active cell growth resulting in their rupture andcell release into the growth medium [5]. These problems wererecently overcome by the successful application of the structuralfibrous network of papaya wood (SFNPW) in a novel procedureof fungal (Aspergillus terreus) hyphae immobilization [6]. Theimmobilization matrix of SFNPW has extensive surface area,depressions and cavities making it ideally suited for immobiliza-tion of microbial cells. Application of this new immobilizationtechnique is reported here for the production of a unique type ofbiosorbent by immobilization of Phanerochaete chrysosporiumonto the SFNPW, for the production of a biosorbent system toremove Zn(II), illustrating its application potential in environ-ment biotechnology for the treatment of wastewaters containingheavy metals.

2. Materials and methods

2.1. Microorganism and culture medium

aboratories Complex, Ferozepur Road, Lahore-54600, Pakistan. Tel.: +92 4223 0688x291/92 92 541 8870; fax: +92 42 923 0705.

E-mail address: iqbalmdr@brain.net.pk (M. Iqbal).P. chrysosporium (ATTC 24725) was maintained by subculturing on potato

dextrose agar slants. Hyphal suspension for immobilization was prepared from

141-0229/$ – see front matter © 2006 Elsevier Inc. All rights reserved.oi:10.1016/j.enzmictec.2006.02.019

M. Iqbal, A. Saeed / Enzyme and Microbial Technology 39 (2006) 996–1001 997

7-day old cultures grown on potato dextrose agar plates at 35 ± 2 ◦C. The liquidgrowth medium consisted of (g/l of distilled water; pH adjusted to 4.5): d-glucose, 10.0; KH2PO4, 2.0; MgSO4·7H2O, 0.5; NH4Cl, 0.1; CaCl2·H2O, 0.1;thiamine, 0.001.

2.2. Immobilizing material and technique

Structural fibrous network of papaya wood (SFNPW) was obtained fromthe felled dried trunk of the matured tree of Carica papaya. The trunk,15–20 cm diameter, appears like a hollow collapsible cylinder. The cylinderwall, 0.5–0.8 cm thick, is a weak woody structure, made up of intertwiningfibrous tissue mass. The outer wood surface is covered by papery bark, whichon peeling exposes a honey beehive like structure (Fig. 1a), constructed by sev-eral fibrous bundles meshed together in easily peelable layers (Fig. 1b). Toobtain the SFNPW, the hollow cylindrical papaya trunk was cut into smallpieces (2 cm × 2 cm), soaked in boiling water for 30 min, thoroughly washedunder tap water, and left for 2–3 h in distilled water, changed three to fourtimes. The washed SFNPW pieces were oven dried at 80 ◦C to constantweight, cooled and kept in desiccator for subsequent use in immobilizationstudies.

For immobilization of fungal biomass onto SFNPW, inoculum was obtainedfrom 7-day old agar plates. The fungal mat was removed and maceratedaseptically in 10 ml sterile liquid medium using a homogeniser. Optical den-sity of the fungus solution was adjusted to 0.5 ± 0.03 at 650 nm wavelengthusing UV/VIS spectrophotometer (Hitachi 220S) to inoculate approximatelythe same amount of mycelial biomass into each flask (Erlenmeyer, 250 ml)containing 100 ml growth medium and four pre-weighed SFNPW pieces. Cul-ture flasks for free fungal growth, with no SFNPW pieces in the medium,served as the controls. The inoculated flasks were incubated at 35 ◦C andshaken at 100 rpm. After 7 days of incubation, both free and the SFNPW-iwowo

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2.3. Biosorption studies

The biosorption of Zn(II) by SFNPW-immobilized biomass of P. chrysospo-rium from aqueous solution was carried out in batch biosorption-equilibriumstudies. Desired concentrations of Zn(II) solution were prepared by diluting1000 ± 2 mg/l standard Zn(II) stock solution (Zn(NO3)2, Merck). pH of thesolution was adjusted to 5.0, using 0.1 M NaOH. Fresh dilutions were used foreach biosorption study. The biosorption capacity of SFNPW-immobilized andfree fungal biomass (100 mg) was determined by contacting 100 ml Zn(II) solu-tions of known concentration (10–500 mg/l) in 250 ml Erlenmeyer flasks. TheZn(II) solution, incubated with the immobilized biosorbent system, was shakenon an orbital shaker at 100 rpm in tightly stopper flasks at 25 ± 2 ◦C. Free fungalbiomass was removed from metal solution by centrifugation at 5000 rpm for5 min, whereas SFNPW-immobilized fungal biomass was separated from thesolution by simple decantation. Residual concentration of Zn(II) in the metalsupernatant solutions was determined using atomic absorption spectrophotome-ter (UNICAM-969). The effect of pH was determined by equilibrating sorptionmixtures at pH values from 2 to 6. Metal-free solution and fungal hyphalbiomass-free metal solution containing only SFNPW of papaya wood blankswere used as the controls.

2.4. Biosorption-desorption cycles

In order to determine the reusability of the SFNPW-immobilized biomassof P. chrysosporium, adsorption-desorption cycles were repeated five timesby using the same immobilized biosorbent system. Desorption of Zn(II) wasdone using 50 mM HCl solution. The SFNPW-immobilized biomass of P.chrysosporium and free fungal biomass loaded with Zn(II) ions were con-tacted with desorption medium at room temperature and agitated on orbitalshaker at 100 rpm for 60 min. The biosorbent was removed and the super-ns

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mmobilized biomass of P. chrysosporium were harvested from the medium,ashed twice with distilled water and stored at 4 ◦C until use. The dry weightf the fungal biomass immobilized within SFNPW was determined as theeight difference of SFNPW before and after fungal growth dried at 70 ◦Cvernight.

ig. 1. Immobilization of P. chrysosporium on the structural fibrous networkf papaya wood (SFNPW): (a) SFNPW piece before immobilization of fungalyphae; (b) single (enlarged) peeled layer of fibrous network showing inter-

persed irregular hollow spaces; (c) SFNPW piece (completely covered) aftermmobilization of fungal hyphae.

os

atant was analysed for Zn(II), released into the solution, by atomic absorptionpectrophotometer.

.5. Reproducibility and data analysis

Unless otherwise indicated, the data shown are the mean values from threeeparate experiments. Statistical analysis of the data was carried out accordingo the Duncan’s new multiple range test [7].

. Results

.1. SFNPW-immobilized biosorbent preparation

Microscopic examination of the SFNPW revealed hyphalrowth within the matrix within 24 h of incubation. Completeoverage of the SFNPW with the hyphae of P. chrysosporiumccurred within 5 days (Fig. 1c). However, biomass accumula-ion/loading was noted to continue until the attainment of sta-ionary phase of growth at day 7. These observations indicate thathe SFNPW-immobilized biosorbent system can be made sim-ly by inoculating fungal hyphae suspension to the liquid growthedium containing the SFNPW without any prior chemical or

hysical treatment. In contrast, production of immobilizationeads from polymeric materials for commercial applications isxpensive, laborious and requires sophisticated equipment. Thecanning electron microscopy of SFNPW-immobilized fungalyphae revealed a uniform growth along the surface of SFNPWndicating that hyphae are not localized in isolated patches butre woven into contiguous mass. This uniform distribution is anmportant criterion for efficient biosorption of heavy metal ionsn the entire surface area of the fungal hyphae. In free growinguspension cultures, the hyphal biomass forms pellets which

998 M. Iqbal, A. Saeed / Enzyme and Microbial Technology 39 (2006) 996–1001

may lead to diffusional restriction thus limiting the availabilityof sites for the biosorption of heavy metal ions.

3.2. Biosorption performance of SFNPW-immobilizedfungal biomass

To demonstrate the metal removal potential of SFNPW-immobilized fungal biomass as a new biosorption system,both free and immobilized fungal hyphal biomass was con-tacted with Zn(II) solution (50 mg/l) in batch experiments. TheZn(II) removal capacity of 100 ± 3.6 mg of the free hyphaebiomass was 20.57 ± 0.9 mg Zn(II)/g dry fungal biomass ascompared with 29.34 ± 0.81 mg/g by 100 ± 3.9 mg of fungalhyphae biomass immobilized onto SFNPW. This indicates a42.63% greater Zn(II) removal by the SFNPW-immobilizedbiosorbent system having the same quantity of biomass thanthe free fungal biomass. The removal of Zn(II) by SFNPWwithout immobilized fungal hyphae in the control run was4.98 ± 0.15 mg/g dry SFNPW. It is not possible to predict howmuch of it contributed to the 29.34 ± 0.81 mg/g Zn(II) biosorbedby the SFNPW-immobilized fungal biosorbent system. Theseresults, however, clearly indicate that the presence of SFNPWas an immobilization matrix has not affected the biosorptioncapacity of immobilized fungal biomass.

The capacity of SFNPW, SFNPW-immobilized and free fun-gal biomass to remove Zn(II) as a function of time is shownin6lpevhr

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Fig. 3. Effect of pH on the sorption capacities of SFNPW, SFNPW-immobilizedand free biomass of P. chrysosporium for Zn(II); 100 ml of 100 mg/l Zn(II)solution was mixed with each biosorbent at 100 rpm and 25 ◦C for 60 min.

at different pH values in the range of 2–6 (Fig. 3). Appropriate pHof the sorption mixture was maintained by adding 0.1 M NaOHor HCl and measured at biosorption equilibrium. Biosorptionof Zn(II) was low at a higher acidic pH of 2. However, Zn(II)sorption capacity of all the three biosorbents increased withincreasing pH, and was maximum at pH of 5. A further increasein Zn(II) sorption between pH 5 and 6 was insignificant. Sincethe optimum pH for Zn(II) biosorption by SFNPW-immobilizedfungal biomass, free fungal biomass and naked SFNPW wasfound to be 5.0, therefore, this pH was used for further study.

3.3. Metal removal capacity of SFNPW-immobilized fungalbiosorbent and adsorption isotherms

Zn(II) uptake was noted to increase with the increase in metalion concentration in the solution until it reached the maximumcapacity of 66.17, 46.62 and 11.21 mg/g biosorbent for SFNPW-immobilized fungal biomass, free fungal biomass and nakedSFNPW, respectively (Fig. 4). This indicated 41.93% greatercapacity of sorption by the SFNPW-immobilized biomass sys-tem than the free hyphae biomass. The relationship between

Fsb

n Fig. 2. Rapid biosorption rates were observed at the begin-ing (first 30 min), which achieved equilibrium after about0 min of biosorbent-metal contact of both free and immobi-ized fungal biomass. This rapid rate of sorption has a significantractical importance for applications in small reactor volumesnsuring efficiency and economy. At each stage of time inter-al, SFNPW-immobilized fungal biomass removed significantlyigher Zn(II) than free fungal biomass, thus showing the supe-iority of SFNPW-immobilized biosorbent system.

The effect of pH on the biosorption of Zn(II) by SFNPW-mmobilized fungal biomass, free fungal biomass and nakedFNPW was determined by equilibrating the sorption mixture

ig. 2. Removal of Zn(II) from 50 mg/l solutions, pH 5.0, by 1 g/l P. chrysospo-ium free or immobilized onto 473 mg/l of structural fibrous network of papayaood (SFNPW) or 473 mg/l of SFNPW as related to time of contact duringrbital shaking at 100 rpm at 25 ◦C.

ig. 4. Effect of metal ion concentration on biosorption of Zn(II) from aqueousolution; 100 ml of Zn(II) solution (10–500 mg/l, pH 5) was mixed with eachiosorbent at 100 rpm and 25 ◦C for 60 min.

M. Iqbal, A. Saeed / Enzyme and Microbial Technology 39 (2006) 996–1001 999

Fig. 5. Langmuir isotherms for Zn(II) sorption by SFNPW, SFNPW-immobilized and free biomass of P. chrysosporium.

metal removing capacity (qeq) and concentration at equilibrium(Ceq) was examined using the Langmuir equation:

qeq = qmaxbCeq

1 + bCeq(1)

where qmax and b are the maximum metal removal capacity(mg/g) and Langmuir constant (l/mg), respectively.

The data fit the Langmuir isotherms model well (Fig. 5).The values of qmax, b and correlation coefficient r2, as calcu-lated from Fig. 5, are presented in Table 1, from which it maybe noted that qmax obtained with SFNPW-immobilized fungalbiomass was appreciably higher in comparison with the qmaxobtained with free biomass. A higher value of b also impliedstronger bonding of Zn(II) to fungal biomass when immobilizedon SFNPW than the fungal biomass present in the free state.

The Langmuir parameters can also be used to predict affinitybetween the sorbate and sorbent using the dimensionless sepa-ration factor RL, which has been defined by Hall et al. [8] as

RL = 1

1 + bC0(2)

where RL is the dimensionless separation factor, C0 the initialconcentration (mg/l) and b is the Langmuir constant (l/mg).

The value of RL can be used to predict whether a sorptionstosff

TLba

B

SFS

Table 2Characteristics of adsorption Langmuir isotherms

Separation factor, RL Type of isotherms

RL > 1 UnfavourableRL = 1 Linear0 > RL < 1 FavourableRL = 0 Irreversible

Fig. 6. Value of separation factor RL, for the sorption of Zn(II) by free orSFNPW-immobilized biomass of P. chrysosporium.

3.4. Distribution coefficient

The adsorption distribution coefficient (K), which is ratioof the equilibrium concentration in solid and aqueous phaseis shown in Fig. 7. A high value of distribution coefficient isthe characteristic of a good biosorbent. SFNPW-immobilizedfungal biomass exhibited a K value of 4134 ml/g dry weightat Ceq of 1.977 mg Zn(II)/l, which is almost 2.6 times higherthan the distribution coefficient value obtained with free fungalbiomass (1558.3 ml/g dry weight at a Ceq of 4.12 mg Zn(II)/l).These results clearly indicate that SFNPW-immobilized fun-gal biomass, in addition to having higher biosorption rate andcapacity (Figs. 2 and 4), also showed higher distribution valuewhich confirms the superiority of SFNPW-immobilized fungalbiomass over free biomass.

Fc

ystem is “favourable” or “unfavourable” in accordance withhe criteria shown in Table 2. The values of RL for sorptionf Zn(II) on free and SFNPW-immobilized fungal biomass arehown in Fig. 6. The RL values indicated that sorption was moreavourable for SFNPW-immobilized fungal biomass than freeungal biomass.

able 1angmuir constants and correlation coefficients for biosorption of Zn(II) ionsy structural fibrous network of papaya wood (SFNPW), SFNPW-immobilizednd free fungal biomass from aqueous solution

iosorbents qmax (mg/g) b (l/mg) r2

FNPW-immobilized biomass 64.08 0.071 0.985ree biomass 44.26 0.039 0.992FNPW 10.83 0.019 0.995

ig. 7. Distribution coefficient of Zn(II) biosorption. Ceq refers to Zn(II) con-

entration at equilibrium.

1000 M. Iqbal, A. Saeed / Enzyme and Microbial Technology 39 (2006) 996–1001

Fig. 8. The performance of SFNPW-immobilized fungal biomass as a Zn(II)biosorbent in five adsorption–desorption cycles.

3.5. Reusability of SFNPW-immobilized fungal biosorbentin repeated adsorption–desorption cycles

Reusability of a biosorbent is of critical significance incommercial applications for metal removal from wastewa-ter. The capacity of the SFNPW-immobilized fungal biosor-bent to adsorb Zn(II) was, therefore, determined by repeatedadsorption–desorption cycles. Higher than 99% desorptionwas obtained after five adsorption–desorption cycles. TheSFNPW-immobilized fungal biosorbent undergoing successiveadsorption–desorption cycles retained good metal adsorptioncapacity even after five cycles (Fig. 8). The total decrease insorption efficiency of SFNPW-immobilized fungal biosorbentafter the five cycles was only about 3.2%, which shows thatSFNPW-immobilized fungal biosorbent has good potential toadsorb metal ions from aqueous solution and can be used repeat-edly.

4. Discussion

An excellent potential of Zn(II) removal from aqueous solu-tion was demonstrated by the SFNPW-immobilized biosor-bent. The maximum Zn(II) biosorption capacity of SFNPW-

immobilized fungal biomass was noted to be 66.17 mg/g,whereas the maximum removal of Zn(II) by free fungal biomasswas 46.62 mg/g. This indicates a 41.63% higher Zn(II) removalby SFNPW-immobilized biosorbent system than the free fun-gal biomass. The maximum adsorption of Zn(II) by SFNPW-immobilized biosorbent was also noted to be higher than theother previously reported biosorbents (Table 3). The removal ofZn(II) by SFNPW without immobilized fungal hyphal biomass(naked SFNPW) in the control run was found to be 11.21 mg/g.Though it is not possible to predict how much of it contributed tothe 66.17 mg/g Zn(II) biosorbed by SFNPW-immobilized fungalbiosorbent system, yet most of it is likely to have been adsorbedon the expanded surface area of this unique biosorbent providedby fungal hyphal biomass immobilized along the outer surfaceof the fibres of SFNPW. From these results, nevertheless, itis clear that the use of SFNPW as an immobilization matrixhas significantly enhanced the biosorption capacity of SFNPW-immobilized fungal biosorbent system and has cause no negativeeffect on the biosorption process. This is a significant achieve-ment in the development of immobilized biosorbent system overthe currently used gel-immobilized biosorbent systems wherea significant decrease in the rate of metal sorption have beenreported in comparison with free cells [18,19]. The reductionin the rate of metal uptake by the gel-immobilized biosorbenthave been projected to be due to limitations in the movement ofmetal ions, or the masking of active sites on the biosorbent [20].Mm

bTsaalafii

Table 3C Phan

B

i (mg

R 10–P 30–S 50–C 6.5A 000S 98.0P 587A 1–A 15–S 10–P

n

omparison between the Zinc(II) removal by SFNPW-immobilized biomass of

iosorbent Operational conditions

pH T (◦C) C

hizopus arrhizus 6–7 n.ahanerochaete chrysosporiuma 7.0 25treptoverticillium cinnamoneum 5.5 28itrobacter strain MCMB-181 6.5 25zolla filiculoides 6.0 18 1argassun sp 4.5 30eat 4.7 25ctivated carbon 4.5 25nimal bones 5.0 20FNPW-immobilized 5.0 25hanerochaete chrysosporium

.a., not available.a Ca-alginate immobilized.

oreover, part of the cell surface might be shielded by the gelatrix and thus would not be available for metal binding [18].A rapid rate of Zn(II) uptake by SFNPW-immobilized fungal

iosorbent system (Fig. 2) is also noted during the present study.his is another advantage of this newly developed biosorbentystem over the other immobilized biosorbent reported in liter-ture. For example yeast cells immobilized in polyvinyl alcoholnd alginate removed copper very slowly and reached at equi-ibrium in 12 and 24 h, respectively [21] which is about 12nd 24 times higher than time taken by SFNPW-immobilizedungal biomass to remove Zn(II) during the present study. Sim-larly, microbial biomass, from activated sludge, immobilizedn sodium alginates took 15 h to reach at equilibrium for the

erochaete chrysosporium (this work) and others found in the literature

qmax (mg/g) Reference

/l) Biomass (g/l)

600 3.0 13.5 [9]600 n.a 39.0 [10]1000 2.0 21.3 [11]8–115 2.0 23.62 [12]

4.0 45.2 [13]n.a 24.35 [14]50.0 11.2 [15]

1000 4.0 31.11 [16]79 4.0 11.55 [17]500 1.0 66.17 This work

M. Iqbal, A. Saeed / Enzyme and Microbial Technology 39 (2006) 996–1001 1001

removal of cadmium(II) which was 100 time higher than the timetaken by the free cells [22]. They suggested that the observedslow rate of cadmium(II) uptake by alginate beads was limitedby diffusion of cadmium(II) through the gel matrix.

Reusability of a sorbent is of crucial importance in industrialpractices for metal removal from wastewater. The study showsthe potential of SFNPW-immobilized fungal biosorbent forremoval and recovery of Zn(II) from contaminated water in fiverepeated cycles without any significant loss in the metal remov-ing efficiency. Furthermore, no significant leakage of entrappedbiomass or physical breakage of SFNPW-immobilized fun-gal biosorbent was observed during these repeated cycles aswas noted with other polymeric matrices immobilized systems[23,12,18], which ultimately resulted in the loss of biosorptioncapacity of these immobilized systems. In conclusion, SFNPWis an inexpensive material, available in all tropical and subtrop-ical countries, and the SFNPW-immobilized fungal biosorbentcan be easily developed by the simple inoculation of culturemedium containing the SFNPW pieces with an appropriate fun-gal hyphal/spore suspension. The innovative immobilizationtechnology developed, thus provides an attractive strategy forthe developing high-affinity biosorption system for the treatmentof wastewater containing heavy metals in low concentration.

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