Effect of pretreatment on Cd 2 + biosorption by mycelial biomass of Pleurotus florida

The effect of pretreatment on the Cd biosorption capacity of mycelial biomass of Pleurotus florida was investigated. For this purpose, the biomass was subjected to physical treatments such as heat, autoclaving and freeze drying and chemical treatments using acids, alkali and organic solvents. All the pretreatment methods improved the biosorption of Cd in comparison with live biomass of P. florida. Among physical treatments, freeze drying showed significant improvement in Cd sorption capacity. Pretreatment of biomass with NaOH showed maximum cadmium biosorption followed by formaldehyde and methanol treatment.


INTRODUCTION
Fungi like other microorganisms can absorb heavy metals from their external environment by means of physico-chemical and biological mechanisms.Biosorption is a process by which living and dead microbial cells are able to remove heavy metal ions from aqueous solutions (Pighi et al., 1989;Siegel et al., 1990).Living cells are likely to be more sensitive to metal ion concentration and adverse operating conditions of pH and temperature.Furthermore, a constant nutrient supply is required for using living cells.Recovery of metals and regeneration of biosorbent is complicated for living cells.In addition, non viable cells frequently exhibits a higher affinity for metal ions compared with viable biomass probably due to absence of competing protons produced during metabolism.So, non viable biomass is preferred than the viable cells.The living cells can be inactivated by physical pretreatments using heat treatment (Galun et al., 1983a;Siegel et al., 1986;Townsley et al., 1986a), autoclaving and vacuum drying (Tobin et al., 1984;Huang et al., 1988a) or treating the biosorbent with chemicals like acids, alkali, detergents, organic solvents (Muzzarelli et al., 1980a, b;Huang et al., 1988a;Azab and Peterson, 1989;Rao et al., 1993;Brady et al., 1994) or by mechanical disruption (Tsezos and Volesky, 1981; Yakubu and *Corresponding author.E-mail: nilanjana00@lycos.com.Dudeney, 1986).These types of pretreat-ments modify the cell surface which is essential for biosorption either by removing or masking the groups or exposing more metal binding sites (Gupta et al., 2000).The aim of this study was to investigate the effect of physical and chemical pretreatment of mycelial biomass of Pleurotus florida on biosorption of Cd 2+ .

Preparation of biomass
Tissues from the fruit bodies of P. Florida were cultured on Potato Dextrose Agar (PDA) media.After 7 days of incubation, the biomass was aseptically transferred into a mineral medium of following composition: glucose (20 g); KH2PO4 (1 g); MgSO4.7H2O(0.5 g); (NH4)2SO4 (0.5 g); distilled water (1000 ml) with a pH of 6.5.The inoculated liquid medium was then agitated in a rotary shaker at room temperature for 7 days at 125 rpm.For the biosorption studies, the mycelial biomass was harvested by filtering the medium through Whatman No. 1 filter paper.Pretreatment with organic solvents: (M10) Treated with 10 ml of 10% (v/v) methanol (CH3OH) and agitated for 3 h.(M11) Boiled for 15 min with 20 ml of 50% (v/v) dimethyl sulfoxide (C2H6SO).(M12) Treated with 100 ml of 10% (v/v) formaldehyde (HCHO) and agitated for 3 h.All the pretreated biomass were washed with distilled water until the pH of the wash solution was in near neutral range (6.8 -7.2) and then subjected to oven drying at 60°C for overnight.

Biosorption studies
Biosorption experiments were carried out using Cd 2+ containing solutions added in the form of Cd(NO3)2.4H2O in distilled water.All the dried biomasses were powdered.Each type of pretreated biomass (0.1 g) was added to 100 ml of 10 mg of Cd 2+ /L at pH 5. The flasks were kept on a rotary shaker at room temperature and 125 rpm.After 2 h of contact time, the biomass was separated by centrifugation of reaction mixture for 15 min at 10,000 rpm and the supernatant were analyzed for metal concentration.

Measurement of metals
Cadmium concentration in the solution was measured by using Varian AA-240 Atomic Absorption spectrophotometer.Before measurement, the solutions containing Cd 2+ were appropriately diluted with deionized water to ensure that the Cd 2+ concentration in the sample was linearly dependent on the absorbance detected.Biosorption experiments were conducted in triplicate and average values were used in the analysis.The amount of Cd 2+ (mg) biosorbed per gram of dried biomass was calculated using the following equation: Where, Q = amount of metal ion biosorbed per gram of biomass (mg g -1 ); Co = initial metal ion concentration (mg L -1 ); C = final metal ion concentration (mg L -1 ); m = weight of biomass in the reaction mixture (g); and V = volume of the reaction mixture.

RESULTS AND DISCUSSION
Pre-treatment of living biomass (M0) using oven drying (M1), Autoclaved (M2) and freeze dried (M3), NaOH (M4), NaHCO 3 (M5), Na 2 CO 3 (M6), Glacial acetic acid (M7), oxalic acid(M8), ortho-phosphoric acid (M9), methanol (M10), dimethyl sulfoxide (M11) and formaldehyde (M12) resulted in an improvement in cadmium biosorption in comparison with living biomass.Figure 1 shows the effect of physical pretreatment of mycelial biomass of P. florida on biosorption of cadmium.It was observed that Q values obtained for all the physically pretreated biomasses were high in comparison with living biomass (from 3.21 to 7.13 mg/g).Freeze dried biomass showed the maximum improvement on cadmium sorption.The bioabsorption capacity of oven dried and autoclaved biomass increased in comparison with live biomass may be attributed to the exposure of latent binding sites.
Figure 2 shows the effect of pretreatment with alkali chemicals.Pretreatment of biomass with NaOH showed maximum increase on biosorption of Cd 2+ by approximately three times in comparison with living biomass (from 3.21 to 9.76 mg/g).In a study by Galun et al. (1987), NaOH treated biomass of Penicillium digitatum also showed enhancement of Cd (II) bioadsorption.Removal of surface impurities, rupture of cell membrane and exposure of available binding sites for metal bioadsorption after pretreatment may be the reason for the increase in metal biosorption.
Figure 3 shows the effect of pretreatment with acids.Oxalic acid and orthophosphoric treatment significantly increased the biosorption of cadmium, which is in agreement with the observation of Huang and Huang (1996) who reported that the acid pretreatment can strongly enhance the adsorption capacity of Aspergillus oryzae.The pretreatment with glacial acetic acid decreased the biosorption of cadmium compared to living biomass which is in agreement with the observation of Kapoor and Viraraghavan (1998) in case of Aspergillus niger.The polymeric structure of biomass surface exhibits a negative charge due to ionization of organic groups and inorganic groups.The higher the biomass electronegativity, greater is the attraction and adsorption of heavy metal cations.Thus, the remaining H + ions on the acid pretreated biomass may change the biomass electro-negativity, resulting in a reduction in bioadsorption capacity.
Figure 4 shows the pretreatment of biomass with organic solvents viz.methanol, dimethyl sulfoxide and formaldehyde which resulted significant improvement in Cd 2+ biosorption.Maximum improvement was noted in case of formaldehyde treatment.Similar response was reported by Kapoor and Viraraghavan (1998).They reported that biosorption of cadmium on A. niger was more efficient after formaldehyde treatment.However, Huang and Huang (1996) suggested that when biomass was pretreated with formaldehyde, methylation of the amino groups present in the cell wall significantly reduced biosorption capacity because the biomass was boiled during formaldehyde treatment.But in the present experiment biomass was simply mixed with formaldehyde, it was not boiled.So the difference in result may be due to the specific treatment.
According to the results of the present experiment, it is obvious that the biomass of P. florida pre-treated physically or chemically is able to remove cadmium ions from aqueous solution.It may be advantageous to use this novel fungal biomass after chemical pretreatment especially with NaOH.Thus the fungal biomass of P. florida may be applied as potent biosorbent for removing cadmium ions from effluents.