Fermentation waste of Aspergillus terreus: a potential copper biosorbent

Ruchi Gulati, R.K. Saxena* and Rani GuptaDepartment of Microbiology, University of Delhi, South campus, Benito Juarez Road, New Delhi 110021, India*Author for correspondence: Fax: +91-11-6885270/6886427, E-mail: micro@dusc.ernet.in

Received 19 June 2001; accepted 29 January 2002

Keywords: A. terreus, biomass, biosorption, copper

Summary

Aspergillus terreus mycelial waste produced during lipase production showed good copper biosorption capacity(160–180 mg Cu2+ biosorbed/g dry biomass). The sorption process followed fast kinetics and the absorptionbehaviour could be explained by a Freundlich isotherm model. The process was temperature independent andunaffected by the presence of many competing ions in a multi-ion situation. Maximum biosorption occurredbetween pH 4 and 5. The biomass could efficiently remove copper from mine effluents. Moreover the loadedbiomass could easily be desorbed by a simple acid wash and could be reused a number of times without a decline inits biosorbing potential, thus making the process cost-effective.

Introduction

Mycelial biomass of most fungi produced in industrialfermentations is disposed off either as landfil or byincineration, thereby posing a potential environmentalhazard. Many scientists have advocated the possible useof this biomass as a potential biosorbent for the removaland recovery of heavy metal ions (Muzarelli et al. 1980;White & Gadd 1990; Wilhelmi & Duncan 1995). It isreported that both living and dead biomass of bacteria,fungi and yeasts is capable of metal accumulation (Gadd1990). The use of dead biomass for metal bindingcircumvents toxicity problems that can occur with livingcells, eliminates the economic component of nutrientsupply and the subsequent metal and biomass recoveryis often easier (Gadd 1990). This process may be a cost-effective alternative for decontamination and/or recoveryof heavy metal ions from metal-contaminated industrialwastes. The fungal biomass contains a relatively highpercentage of cell wall material, which shows excellentmetal-binding capacity (Rosenberger 1975). Luef et al.(1991) used waste biomass of citric acid-producingAspergillus niger, penicillin-producing Penicillium chrys-ogenum and ergotamine-producing Claviceps paspali forthe biosorption of zinc. Waste yeast from a Canadianbrewery was capable of binding 70 mg Cd2+/g dry wt ofbiomass as reported by Volesky et al. (1993).

We have earlier reported excellent bio-indicatingpotential of waste of biomass of A. terreus (obtainedas a byproduct from the fermentative production of theenzyme lipase) for detection of copper in effluentsamples (Gulati et al. 1999a). In an extension of the

same work, we hereby report detailed studies on the useof waste biomass of A. terreus as an efficient copperbiosorbent.

Materials and methods

Organism and growth conditions

Aspergillus terreus, a natural isolate and a potentiallipase producer, was grown in a 20 l fermentor (NBS,USA) containing 5.0 l of lipase production medium at37 �C and pH 9.0 (Gulati et al. 1999b). Approximately300 g (wet weight) of mycelium was obtained from theculture broth of one fermentation run by filtrationthrough Whatman no. 1 filter paper using a vacuumsystem. The biomass so obtained was used for biosorp-tion studies.

Preparation of the biomass for biosorption

The fungal biomass was autoclaved at 121 �C for30 min and then homogenized (Homogenizer fromRemi motors, Mumbai) for 20 min. The homogenizedmass was centrifuged to remove the unwanted intracel-lular material and washed twice with distilled water. Thebiomass thus obtained was dried at 80 �C for 24 h andpowdered using a domestic mixer grinder (Sumeet IndiaLtd., Mumbai, India). This dead and powdered biomass(mostly mycelial walls) was used for carrying outbiosorption experiments.

World Journal of Microbiology & Biotechnology 18: 397–401, 2002. 397� 2002 Kluwer Academic Publishers. Printed in the Netherlands.

Analytical method

The desired concentrations of Cu2+ (10–100 mg/l) wereprepared by dissolving copper sulphate pentahydrate indouble distilled water. The metal sorption experimentswere performed in 250 ml Erlenmyer flasks containing50 ml of the salt solution having the desired concentra-tion of copper at 25 �C for 15 min in a shaker incubator(NBS, USA) at 100 rev/min. In all experiments, exceptfor the effect of biomass concentration, the biomass waskept constant at 0.1 mg/ml of the metal ion solution.Biomass separation from the metal-bearing solution wasachieved by filtration through Whatman filter paper no.1. The filtrates and the biomass-free metal blanks wereappropriately diluted and the residual and initial metalconcentrations were estimated by atomic absorptionspectrophotometer (Shimadzu AA-260) at 324.5 nmwave length (slit width 3.8 A). All the experiments werecarried out in triplicate and repeated twice. The stan-dard deviations are mentioned at appropriate places.

The metal uptake capacity in mg/g (q) was calculatedfrom the initial concentration (Ci) and the final/residualconcentration (Cf) of the metal according to theequation

q ¼ V ðCf � CIÞ=M ðSar et al: 1999Þ:

where, V is liquid sample volume and M is the biomassdry weight. The biosorptive metal uptake was evaluatedstatistically and expressed by the use of a Freundlichadsorption model. The validity of the biosorption modelwas tested at pH 4.0 using two biomass concentrationsi.e. 0.1 mg dry wt/ml and 0.2 dry wt/ml.

The general form of the model is: q ¼ kC1=n which canbe linearized by taking the natural logarithm in the formof ln q ¼ ln k þ 1=n lnC ¼ equilibrium (final/residual)concentration. The intercept ln k gives a measure of theadsorbent capacity and the slope 1/n gives the intensityof adsorption.

Factors affecting biosorption

Different physical factors were studied for their effect onbiosorption. These were:

Time course of copper biosorptionBiosorption of copper (50 mg/l) was studied from aperiod from 5 min to 60 min at 25 �C.

pHThe pH of the metal solution was adjusted to valuesbetween 2 and 5 using 1 M HCl or 1 M NaOH forstudying its effect on biosorption. Biosorption of coppercould not be carried out at pH values above 5.0, due toprecipitation of the metal.

TemperatureAdsorption of copper was carried out at temperaturesranging from 25 to 50 �C for 15 min.

Anions and cationsEffect of metal anions as ammonium salts, viz. chloride(Cl)), sulphate (SO2�

4 ), phosphate (PO3�4 ) and nitrate

(NO�3 ) in the concentration range 0–5 mM and cations

viz. magnesium (Mg2+) and calcium (Ca2+) as sulphateand chloride salts respectively, in the concentration range0–20 mM, were studied on copper biosorption at 25 �Cand pH 4.0. Copper adsorption was also studied in amultimetal situation containing equimolar concentra-tions (50 mg/l) of threemetals viz. Cu2+(CuSO4 Æ 5H2O),Co2+(CoCl2 Æ 6H2O) and Zn2+(ZnSO4 Æ 7H2O).

Removal of copper from mine effluents

The ability of A. terreus biomass to remove copper fromGhatshila copper mine effluents (Bihar, India) wasinvestigated at various concentrations of the biomasswith 10 ml of the effluent sample under the sameexperimental conditions as mentioned above.

Desorption

Elution of copper from the loaded biomass was achievedusing citrate buffer (0.1 M, pH 2.0). The desorbingsolution was dispensed in varying amounts of the bufferviz. 25, 10, 5, 2.5 and 1.0 ml and 5 mg of the loadedbiomass was suspended in it to give solid–liquid (S/L)ratios of 0.2, 0.5, 1.0, 2.0 and 5.0 respectively. Flaskswere incubated for 5 min under shaking conditions(100 rev/min) at 25 ± 2 �C. The amount of metaldesorbed was determined by atomic adsorption spec-trophotometry.

Resorption

The desorbed biomass was subjected to three cycles ofdesorption–resorption. Biosorption/resorption was car-ried out with 50 mg/l copper solution under the sameconditions as mentioned above. Desorption was carriedout using S/L ratio of 1.0 for 5 min after each cycle ofsorption. Each cycle was accompanied by intermittenttap water washings to recharge the biomass afterdesorption.

Results and discussion

Mycelial waste of A. terreus obtained as a fermentativebyproduct after lipase production showed good copperbiosorption capacity (160–180 mg/g biomass). Morethan 90% of the biosorption was complete within5 min and reached an equilibrium within 15 min ofcontact with the metal-bearing solution. However,binding of copper by Cunninghamella blakesleeanareached its peak only after 2 h as reported by Ven-kateswarlu & Stotzky (1989). Brady & Duncan (1994)showed that the process of copper adsorption consistedof two phases, a fast initial phase (within the first fewminutes) and a slow secondary phase in Saccharomyces

398 R. Gulati et al.

cerevisiae. Our process is very fast in comparison to thereported literature.

The adsorption behavior followed a Freundlich iso-therm model. A log–log plot between the residualcopper concentration in the solution and the amountbound to the biomass followed a straight line at twovarying concentrations of biomass i.e. 0.1 and 0.2 mgdry wt/ml (Figure 1). The correlation coefficient be-tween the residual copper and the biosorption capacitywas found to be high i.e. 0.92 and 0.95 for 0.1 and0.2 mg dry wt/ml biomass respectively.

Adsorption of copper ions was found to be unaffectedby temperature from 25 to 50 �C. However it wasaffected by pH. Maximum biosorption of copper oc-curred between pH 4 and 5 (Figure 2). No biosorptionoccurred at pH 2.0. At this pH, almost all the metal ionsare ionized and the carboxylate groups of the biomassdissociate generating the negatively charged surface. The

electrostatic interaction between the ionic species maythus be responsible for metal binding. However at lowpH, the lack of biosorption could be attributed to thepresence of large amounts of protons, which tend tocompete with the metal ions for binding to the cell wall,as reported by Paknikar et al. (1993).

Increasing biomass concentrations caused a decline inthe specific uptake of copper and in the total metal ionsabsorbed (Table 1). There was however an increasedoverall percent removal. This is in agreement with theresults of deRome & Gadd (1987), where the amount ofcopper biosorbed per unit weight of the biomassdecreased with an increase in the concentration ofRhizopus arrhizus, Cladosporium resinae and P. italicum.The dependence of adsorption on cell density is ex-plained by the fact that increase in cell density reducescell distances and thus less sites are available for metalbinding (Itoh et al. 1975; Brady & Duncan 1993).

Generally, metal biosorption studies are carried outwith solutions containing a single metal ion. Suchstudies are important but the validity of this applicationis in question when dealing with actual effluents. Thesemight contain competing ions, which may interfere withrecovery of the test metal (Singleton & Simmons 1996).Accordingly, the effect on copper biosorption of variouscations and anions at different concentrations wasstudied. Results showed that copper biosorption wasnot affected by Mg2+ ions. Only a 20–25% decline inbiosorption was observed in the presence of Ca2+ ions(Figure 3). Further copper biosorption by A. terreus wasunaffected in presence of NO3), PO3�

4 and Cl) ions andshowed a feeble decline with SO2�

4 ions (Figure 4). Incontrast to our studies, uptake of copper was found tobe reduced in presence of Mg2+ and Ca2+ in case ofNeurospora crassa (Somers 1963). Similarly copperbinding in P. spinulosum was reduced by Mg2+, Co2+

and Zn2+ (Townsley & Ross 1985). However, uptake ofCu2+, Cr3+ and Zn2+ was not affected by Ca2+ in caseof Phormidium laminosum (Sampedro et al. 1995). Zhou& Kiff (1991) however reported inhibition of Cu2+

biosorption by sulphate in R. arrhizus.Besides copper, A. terreus biomass is able to adsorb

Zn2+ and Co2+, with biosorption capacities 52.0 and57.0 mg/g for Zn2+ and Co2+ respectively. The effect ofthese competing metals in a multimetal situation showedthat Cu2+ biosorption was unaffected by the presence ofother metals (Table 2). This shows that binding sites ofCu2+ are distinct from Zn2+ and Co2+. The capacities

Figure 1. Freundlich adsorption isotherm for copper biosorption at

0.1 (n) and 0.2 (m) mg dry wt/ml biomass concentrations (pH 4.0).

Biosorption was carried out at various concentrations of copper (10–

100 mg/l) at 100 rev/min and 25 �C for 30 min.

Figure 2. Effect of pH on copper biosorption by A. terreus biomass.

Biosorption was carried out with 50 mg/l copper at 100 rev/min and

25 �C for 15 min.

Table 1. Effect of varying concentrations of biomass on copper

biosorption by A. terreus.

Biomass

concentration (lg/ml)

Mg Cu2+ biosorbed/g

dry wta

40 377.5 ± 10.6

100 156.0 ± 26.2

200 103.0 ± 2.4

a Biosorption was carried out at 25 �C for 15 min at 100 rev/min

and pH 4.0 with 50 lg copper/l.

Aspergillus terreus as copper biosorbent 399

of binding of these metals followed the order Cu2þ >Zn2þ > Cd2þ > Co2þ (Mattuschka et al. 1993). In con-trast to this, sorption followed the order Zn2+ and Co2+

could be common or the biomass might have a higheraffinity for Zn2+ than for Co2+.Aspergillus terreus biomass was also investigated for

its potential to remove Cu2+ from the effluent of theGhatshila copper mines (Bihar, India) containing 69–71.5 mg Cu2+/l at pH 4.2. Experimental results showed

that the biomass could adsorb about 119.4 mg/g dry wt.of Cu2+ at 1 mg/10 ml biomass concentration (Ta-ble 3). Specific uptake decreased with increasing bio-mass, but increased the percent removal.

For biosorptive removal of metals to be economicallyviable, regeneration of the biomass and its subsequentreuse is essential. We had earlier reported that citratebuffer was the best eluent for desorption of copper fromthe loaded biomass (Gulati et al. 1999a). To check forthe efficiency of the eluent, desorption of copper wascarried out using different S/L ratios. It was observedthat desorption was achieved in very short periods oftime (5 min) using 0.33 M citrate buffer (pH 2.0) with avery high S/L ratio of 5.0 and metal recovery of 75.5%(Table 4). Recovery could be further improved to 90–92% by slightly decreasing the S/L ratio. However tocheck for maximum elution, desorption was carried outfor a time period of 20 min. No further change indesorption capacity was reported on extending the timeperiod. Adsorption–desorption was observed for threecycles without reduction in the adsorption capacities

Table 2. Biosorption of heavy metals in a multi-ion situation by

A. terreus biomass.

Metals in

solution

Mg metal biosorbed/g dry biomassa

Copper Zinc Cobalt

Cu2+ 161.2 ± 2.5 – –

Zn2+ – 52.2 ± 10.6 –

Co2+ – – 57.0 ± 9.5

Cu2+ + Zn2+ 160.7 ± 1.2 40.2 ± 3.2 –

Cu2+ + Co2+ 162.4 ± 2.6 – 41.6 ± 6.6

Cu2+ + Co2+ + Zn2+ 154.6 ± 1.9 42.4 ± 9.5 32.4 ± 8.3

a Biosorption was carried out at 25 �C for 15 min at 100 rev/min

and pH 4.0 with 50 mg/l of each metal.

Figure 3. Effect of different cations at a concentration of 100 and

200 mM on copper biosorption by A. terreus biomass.

Figure 4. Effect of various anions at a concentration of 1, 2 and 5 mM

on copper biosorption by A. terreus biomass.

Table 3. Removal of copper by A. terreus biomass from a sample of

Ghatshila copper mine effluent.

Amount of biomass

added to 10 ml

sample (mg)

Mg Cu2+

biosorbed/g

dry wta

Percentage of

removal

1 119.0 ± 10.6 16.7

2 74.3 ± 4.6 20.8

5 40.1 ± 9.4 28.1

a Biosorption was carried out at 25 �C for 15 min at 100 rev/min

with 10 ml of the effluent sample.

Table 4. Desorption of copper from A. terreus biomass using citrate

buffer as eluent at various S/L ratios.

Volume of

eluent (ml)

S/L ratios Percentage of

desorption in 5 min

25.0 0.2 91.4 ± 2.5

10.0 0.5 93.6 ± 7.0

5.0 1.0 85.4 ± 1.0

2.5 2.0 82.9 ± 10.2

1.0 5.0 75.5 ± 2.5

S – dried waste biomass (lg); L – eluent (ml).

Table 5. Desorption–resorption of copper for three cycles by waste

biomass of A. terreus.

Cycle no. Biosorption/

resorptionaPercentage

of desorption

I 168.4 ± 3.0 84.3

II 170.3 ± 10.6 87.9

III 162.3 ± 1.1 88.5

Each cycle was accompanied by intermittent washings with tap

water to recharge the biomass.a Biosorption was carried out for 15 min and desorption for 5 min.

400 R. Gulati et al.

(Table 5), as the biomass could be simply rechargedusing tap water after each cycle of desorption.

Thus, from the foregoing discussion it can be con-cluded that waste biomass of A. terreus has the potentialto efficiently remove copper from mine effluents. Theprocess is unaffected by various physiological conditionsand maintains its high level of biosorption capacitybesides being highly cost-effective and industrially fea-sible due to reusability of the biomass after desorption.Thus, the large amounts of mycelium produced duringthe production of the industrially important enzymelipase can be efficiently put to use for recovery of metalsfrom mines and waste waters.

Acknowledgements

The authors wish to thank Ms Rekha Kohli and MsMamta Samtani for their assistance in preparation ofthis manuscript. Ruchi Gulati acknowledges the finan-cial assistance given by the University Grants Commis-sion and the help provided by Dr Dinesh and Mr Kishenat University Science Instrumentation Centre in usingthe atomic absorption spectrophotometer.

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