Abstract

Biosorption, in which viable or nonviable biomass, is directed to accumulate toxic heavy metals from aqueous stream is attracting wide attention as an alternate wastewater treatment technology.The present study reports screening and evaluation of nonedible woodrotting macro fungi (mushrooms), which occur abundantly in tropical forests, for metal binding potential. Among the nine mushrooms screened for copper (II) uptake, Ganoderma lucidum exhibited the highest binding potential. No specific pretreatment was required for its potential application. Simple gravity settling could be used for separating the sorbent from the aqueous stream. G. lucidum could bind many other metallic cations. Systematic study on Cu and La indicated ion exchange/ complexation to be the major mechanism for metal sorption. Sorbed metals could be completely recovered by mineral acid or EDTA treatments. Biosorbent was used in continuos flow column reactor for mixed rare earth metals removal. Sorbent could be used in repeated cycle of sorption after desorbing the metal with dilute acid. Recovery of sorbed metal in concentrated form was possible.

Introduction

Biosorption, where microrganisms are used for metal sequestration, is emerging as an alternate technology for the removal/recovery of toxic and precious metals from wastewater streams. Extensive literature is available on metal uptake potential of various microbial biomass (Voloesky, 1990; Bailey et al. 1999). Most of these studies have been either with laboratory grown or waste biomass generated from other industries. They generally require immobilization, chemical pretreatment or energy intensive separation processes for separating metal laden sorbent from the aqueous phase. Recently studies, on the use of abundantly occurring marine algae as biosorbent, have been reported (Figueira et al. 2000). Although macrofungi (Mushrooms) are widely occuring and seem to possess physical characteristics required for the use in continuos flow reactors, metal binding potential of these biomass have been rarely investigated. In this communication, results on the screening of few mushrooms for metal uptake and the evaluation of Ganoderma lucidum as biosorbent is reported.

Materials and Methods

Preparation of sorbent: Nonedible mushrooms were collected during post monsoon period from forests and plantations in Kerala, India. Sorbents were prepared by pulverising the sun dried fruiting bodies in a hand grinder. 600-1200 mm particles were used as the biosorbent. Identification of mushrooms was done by Royal Botanical Garden, Kew, UK.

Adsorption experiments: Screening of the mushrooms for metal sorption potential was carried out using Cu (II). Adsorption studies were carried out with 100 ml adsorbate solution adjusted to pH 4.0 ± 0.2 with 0.1 M acetate buffer and 250 mg sorbent. Mixture was agitated in end-on-end rotatory shaker. Cu (II) concentration of 1mM was maintained for kinetic studies whereas 0.2 –2.0 mM range was used for equilibrium uptake studies. After required contact time, variable for kinetic studies and 3h for equilibrium uptake, Cu (II) was estimated in the supernatant after separating the sorbent by gravity settling. Similar procedure was used for other metallic cations and Ca + La binary metal system.

Pretreatment of sorbents: Three procedures were followed for the pretreatment of the sorbent. HNO₃-HCHO and H₂SO₄–HCHO treatments were carried out as per the procedure described by Freer et al (1989). 10g of G.lucidum was mixed with 150ml of 3% HNO₃ and 0.25ml 35% HCHO and was kept in boiling waterbath for 15 min. 20g of G. lucidum was mixed with 200ml 0f 0.2M H₂SO₄ and 10ml 35% HCHO. Mixture was agitated at 100 rpm in a rotatory shaker at 50 ⁰C for 2h. Hot alkali treatment was carried out by suspending 20g sorbent in 40 % NaOH. The mixture was held at 120 ⁰C for 4h (Muzzarelli et al. 1980). After each of these treatments, residue was separated by gravity settling. Treated sorbent was washed several times with distilled water to remove acid/alkali and dried at 40 ⁰C at 24h before using for adsorption studies.

Effect of soluble ligands on Cu(II) Uptake : Effect of soluble complexing ligands on Cu (II) uptake by G. lucidum was studied at 1mM sorbate and 1 and 10mM ligand concentrations. Adsorption experiments were carried out under similar conditions as described earlier. After 3h contact time, the sorbent was separated and Cu (II) was determined in the supernatant.

Batch desorption studies : Adsorption of Cu (II) onto the sorbent was carried out using the procedure described earlier. After separating the supernatant, sorbent was suspended into 10 ml of desired regenerant and agitated for 3h. Desorbing agents used in the present study were 0.1 M HCl, 1 M EDTA, 0.1 M CaCl₂ and distilled water adjusted to pH 4.0 with 0.1 M acetate buffer.

Column reactor studies with mixed rare earths: Column reactor study was carried out with mixed rare earth metals, which was procured from Indian Rare Earth’s Ltd, Kerala, India. Composition of the rare earths as provided by the manufacturer consisted of La 23; CE 46; Nd 5 ; Pr 20 and Sm. 4 and rest of the rare earth 2% by weight. A Plexiglas column of 50 mm diameter with 600 mm sorbent bed depth was used for study. Mixed rare earth’s solution, at a concentration of 250 mg/l (as RCl₃) and buffered to pH 4.0 ± 0.2, was passed through the column at a flow rate of 1.018m³/m²/h. After sorbent exhaustion, it was regenerated with 0.1 N HCl, washed with 2 bed volumes of distilled water and second adsorption cycle was started.

Analytical and Instrumentation Procedures

All metal ions were determined using AAS procedure except for rare earth’s and uranium, which were analysed as per the procedures of Onishi and Skein (1972) and Summate (1980) respectively.

EPR spectra were recorded with E-106 EPR spectrophotometer (Varian). EDAX analyses were conducted using Kevex apparatus attached to the SEM (Jeol, Japan).

Results and Discussion

Screening of Macrofungi : Nine different nonedible macrofungal species (mushroom) were screened for Cu (II) Uptake. Batch equilibrium studies were conducted with each of these biomass, which followed a typical saturation profile. Langmuir relationship, which has been extensively used for the comparison of the metal uptake capacities of biosorbents, was used in this study in the following lineraised form

C_e/q_e= C_e/Q_max+ 1/b Q_max

Where q_e and Q_max are Cu (II) sorbed at equilibrium concentration of C_e and maximum uptake capacity in mmoles per unit weight of sorbent respectively, b is a constant.

Results presented in Table 1 indicate that Q_max varied from 0.048 to 0.38 mmol/g sorbent. G. lucidum exhibited the maximum uptake potential which was higher than few biosorbents reported in literature (Muzzarell et al.1980; Tobin et al.,1984). All further studies were carried out with G. lucidum.

Table 1 Q_max of different fungal species

Species	Q_max (mmol/g)
Coriolopis strumosa	0.115
Daedalea tenuis	0.109
Lentinus strigosus	0.167
Lenzites malaccenis	0.143
Phellinus xeranticus	0.173
Rigidoporus lineatus	0.173
Rigidoporus microporus	0.104
Trametes lactenia	0.048
Ganoderma lucidum	0.375
Filtrasorb-400	0.030

Pretreatment of the sorbent: Pretreatment is mainly aimed at enhancing the metal binding potential, improving the stability and physical characterstics of the biosorbent required for the field application. Batch kinetic and equilibrium studies were conducted after HNO₃-HCHO, H₂SO₄-HCHO and alkali treatments. Apparent adsorption rate (k) was calculated from the kinetic data by plotting metal sorbed per unit weight of the sorbent versus t ½. A compilation of Q_max and k for native and treated G. lucidum is given in Table 2. Although there was not much change in apparent rate after pretreatments, increase in Q_max was observed.

Table 2 k, Q_max and Yield after Different pretreatments

Treatment	k	Q_max	Yield
Native	0.062	0.383	1.00
HNO₃+ HCHO	0.070	0.425	0.79
H₂SO₄ + HCHO	0.070	0.332	0.84
Alkali	0.061	0.478	0.65

When the yield after pretreatment was taken into consideration. Q_max. was marginally reduced. However, this observation is significant from the application point of view, as the reactor volume can be reduced with these treated sorbents.

Mechanism of metal uptake by G. lucidum: EPR, EDAX and quantification of released H⁺ and metallic cations during sorption were used to get an insight into the mechanism of metal binding. EPR spectrum of Cu (II) biosorbent was indicative of square planar geometry of Cu (II) with a Co-ordination number of four. EDAX of the sorbent before and after metal sorption showed that calcium ions of the sorbent was replaced by Cu (II). 0.3 mmol Ca (II) and 0.048 mmol H⁺ions were released per unit weight of the sorbent as against 0.383 mmol Cu (II) sorbed. These observations suggest that ion excahnge/ complexation to be the major mechanism for metal binding by G. lucidum, similar to other biosorbents (Treen sears et al., 1984; Fourest and Roux, 1994).

Effect of anionic ligands on Cu (II) uptake by G. lucidum: Soluble anionic ligands are often present along with the metals in industrial wastewaters and profoundly affect the metal binding by the biosorbent. In this study, effect of few such ligands on Cu (II) binding by G. lucidum was determined at 1 and 10 mM ligand concentrations with Cu (II) concentration being maintained at 1 mM. Significant decrease was observed with cyanide and EDTA. Effect of other ligands could be represented by the following sequence. Perchlorate> Tartrate> Citrate> Carbonate> Nitrite > acetate. Results with few of these ligands are given in Fig 1

Uptake of other metallic cations by G. lucidum : Affinity of biosorbent for other metallic cations was evaluated. Results presented in Table 3 show that G. lucidum can bind to all metal ions used in the present study although there was variation in the uptake capacity. This observation is similar to R arrhizus and A. niger, which can also bind a wide range of metallic cations (Muzzerelli et al. 1980; Tobin et al.1984). When Cu (II) and La (III) ions were present together, La (III) was preferentially taken up (data not shown). This indicates that probably oxygen rich functional groups of the sorbent are involved in metal binding.

Table 3 Q_max of G. lucidum for metal Cations

Metal	Q_max mmol/g
Zn	0.31
Cd	0.26
Ni	0.29
Cu	0.38
Cr	0.33
La	0.36
U	0.34

Desorption Studies: Among the four regenerants used for recovering the sorbed metal ions from the sorbent. Results showed that complete desorption was possible with both EDTA and HCl, whereas only 30% could be recovered with CaCl₂. Desorption with distilled water adjusted to pH 4.0 was negligble. After desorption with 0.1N HCl,the material was washed with distilled water and was reused for subsequent sorption cycle in batch mode. Results indicated that there was no significant loss in metal sorption capacity upto three cycles.

Column Studies for removal of rare earth elements from aqueous stream : Processing of the monazite ore, which is the most abundant source of rare earths in India, generates a large volume of wastewater having rare earth and thorium. To study the feasibility of the field

Fig 2 Regeneration and Reuse of Biosorbent for Mixed Rare Earth

application of biosorbent, performance of the fixed bed reactor configuration was evaluated with mixed rare earths as influent. Rare Earth solution (250 mg/l as RCl₃) buffered to pH 4.0 was passed through the sorbent column in down flow mode. The run was continued until biosorbent was completely exhausted. The exhausted sorbent bed was regenerated with 0.1N HCl for recovering sorbed rare earths. Second sorption cycle was terminated when eluate concentration reached 1/10^th the influent concentration. From the results given in Fig 2, it could be observed that there was no significant loss in capacity in two cycles. Sorbed rare earths could be recovered as concentrated 40x solution.

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