Fungal isolate (Eup. ludwigii) was isolated from a soil located in Shebein El-koom, Menoufia Governorate, Egypt, and identified by Regional Center for Mycology and Biotechnology (Al-Azhar University, Cairo, Egypt). The medium used for the growth of fungus was Potato Dextrose (PD) medium (Oxoid, England). For production of the biomass, the fungus was cultivated on a rotary shaker (Jeio Tech SI-900 R, Korea) at 125 rpm and 28˚C for 3 days in 500 ml Erlenmeyer flask containing 250 ml of PD medium.

After 3 days of growth, the fungal biomass was harvested using a plastic sieve, followed by washing with generous amounts of double distilled water (ddH₂O) to remove residual growth medium, and drained to remove excess water by gentle pressing through Whatman filter paper No. 1. This biomass will be referred to as untreated biomass.

According to Gharieb et al. [9] with some modifications, NaOH treatment was performed by boiling viable fungal biomass in 0.5 N NaOH (1:10, w/v) for 15 min. The resulting biomass was washed extensively with ddH₂O until the pH of the washing solution was close to neutral range (6.8 - 7.2). This biomass will be referred to as treated biomass. Both untreated and NaOH-treated biomasses were dried in an oven for 24 h at 60˚C. Then, dried biomasses were powdered in a mortar with a pestle, sieved through a sieve with 125 μm openings, and stored in a desiccator for future use.

All chemicals used in the present study were of analytical grade. Stock metal solutions of Ni²⁺ and Cd²⁺ (1000 mg∙L⁻¹) were prepared separately by dissolving NiSO₄.6H₂O and 3CdSO₄∙8H₂O, respectively in ddH₂O. For experiments with various metal concentrations, the stock solutions were diluted further with ddH₂O. The pH value of each test metal solution was adjusted to desirable value with 0.1 M HCl or 0.1 M NaOH.

The biosorption of Ni²⁺ and Cd²⁺ from aqueous solutions were carried out in batch systems. 0.04 g of dried fungal biomass (untreated or NaOH-treated) was added to 100 ml Erlenmeyer flasks containing 20 ml metal solution with concentration of 50 mg∙L⁻¹, and agitated in an orbital shaker (Jeio Tech SI-900 R, Korea) for 120 min at 125 rpm and 25˚C. The effect of initial pH (1 - 8), time contact (15 - 120 min), initial metal ion concentration (50 - 400 mg∙L⁻¹), temperature (15 - 40˚C), biomass dose (0.5 - 5 g∙L⁻¹), and shaking rate (0 - 200 rpm) on the metals biosorption were studied. After the end of each experiment, the mixtures were centrifuged (for 5 min at 10000 rpm) and metal ion concentrations in the supernatant were determined. All biosorption experiments were done in triplicate and the mean values were reported. The amount of adsorbed metal ions is estimated as the amount of metal (mg) per unit of biomass dry weight (g) using the following equation:

where V is the volume of metal solution (L), C_i is the initial metal concentration (mg∙L⁻¹), C_f is the final/residual concentration (mg∙L⁻¹) and M is the amount of biomass (g). The percent biosorption of metal ion was calculated as follows:

Following the metal biosorption experiments, metal-loaded biomasses were separated by centrifugation, washed, and contacted with 20 ml of various elutants (at solid/liquid ratio 0.1 g 20 ml⁻¹) for 20 min on an orbital rotary shaker set at 125 rpm at 25˚C ± 1˚C. The elutants used were 0.1 M HCl, 0.1 M CaCl₂, 0.1 M Na₂CO₃, 0.1 M EDTA, and ddH₂O. The biomass was separated from elutants by filtering the reaction mixture through 0.45 μm filter paper and the filtrate was analyzed for metal concentration to investigate the desorption efficiency. The desorption efficiency was calculated from the amount of metal ions adsorbed on the biomass and the final metal ion concentration in the biosorption medium using the following equation:

For the regeneration of biomass eluted using the most efficient elutant, a generous amount of ddH₂O was used to rinse the regenerated biomass till pH in the solution reached the range of 6.8 to 7.2. Then, biomasses were dried at 60˚C for 48 h, and then re-suspended in metal containing solutions for the next biosorption cycle and this biosorption-desorption cycle was repeated five times.

The concentrations of Ni²⁺, Cd²⁺, Ca²⁺, and Mg²⁺ ions in the supernatant of biosorption medium were determined by using an atomic absorption spectrophotometer (AAS) (Unicam 929, Philips Company, UK) with an air-acetylene flame. The instrument calibration was checked periodically by using standard metal solutions. The concentrations of Na⁺ and K⁺ ions in each sample filtrate from the experiments for ion exchange study were determined by using a flame photometer (JENWAY, UK) with reference to appropriate standard solutions.

Several mathematical models have been developed to quantitatively express the relationship between the extent of adsorption and the residual solute concentration. The most widely used models are the Langmuir and Freundlich adsorption isotherm models [14] . To determine the adsorptive capacity of Eup. ludwigii for Ni²⁺ and Cd²⁺ ions, the initial metal concentration varied from 50 - 400 mg∙L⁻¹; while the biosorbent was constant at 0.04 g 20 ml⁻¹. A Langmuir isotherm was then obtained by plotting the values of biosorption capacity (q) versus the residual metal concentration (C_f) in solution. The classical Langmuir equation is given as follows [15] :

where,

q_e = metal adsorbed on the biosorbent (mg∙g⁻¹) at equilibrium;

q_max = maximum possible amount of metal adsorbed per unit weight of biosorbent;

C_f = residual concentration of metal (mg∙L⁻¹) in the solution;

b = equilibrium constant related to the affinity of the binding sites for the metals.

Equation (4) can be linearized as follows:

When 1/q_e is plotted against 1/C_f, a straight line with slope 1/q_max b is obtained and the intercept is corresponding to 1/q_max. Also, q_max and b were determined. The classical Freundlich equation is given as follows [15] :

where,

q_e = metal adsorbed on the biosorbent (mg∙g⁻¹) at equilibrium;

C_f = residual concentration of metal (mg∙L⁻¹) in the solution;

K_f = an empirical constant that provides an indication of the intensity of adsorption;

n = Freundlich adsorption constant.

This equation can be linearized by taking natural logarithm of both sides of the equation, which can be given as follows:

when the values of log C_f are plotted against the values of log q_e, the adsorption constants (K_f and n) were obtained.

The values presented in the study were means of three replicates and expressed as means ± standard error (SE). Statistical analysis was performed using Statistical Package for the Social Sciences (SPSS) 9.05 for Windows where it was possible to evaluate whether the effect and the interaction among the investigated factors were significant with respect to the standard error.

In order to generate anionic sites without significant modification of the fungal cell wall structure, the biomass of Eup. ludwigii was treated with NaOH. Figure 1 shows the metal biosorption values obtained by untreated and NaOH-treated biomasses. As seen in Figure 1, the treatment of Eup. ludwigii with NaOH significantly enhanced the biosorption capacity from 5.24 ± 0.19 to 12.11 ± 0.28 mg∙g⁻¹ for Ni²⁺ and from 13.34 ± 0.31 to 22.15 ± 0.39 mg∙g⁻¹ for Cd²⁺. An enhanced biosorption capacity as a result of NaOH treatment was also observed by many researchers [9] [16] -[20] . Opposite result was reported by Li and Yuan [21] . They found that pretreatment with NaOH decreased the cadmium biosorption by Rhodotorula sp. Y11. Enhancement of biosorption capacity after NaOH treatment may be due to the exposing of active metal-binding sites embedded in the cell wall and causing availability of more anionic sites [17] . Also, alkaline treatment can activate the hydroxyl groups in fungal mycelium and therefore the epichlorohydrin will be easily introduced [22] .

pH of the aqueous solution plays a vital role in the biosorption process, because it affects the chemistry of metals, and the surface charge and ionization of the functional groups on the fungal cell wall during reaction [23] . The effect of pH solution on Ni²⁺ and Cd²⁺ ions biosorption was carried out in the range of pH 1 - 8 at 50 mg∙L⁻¹ of metal ions concentration. From the results illustrated in Figure 2, it was found that the biosorption capacities of Ni²⁺ and Cd²⁺ ions were lower at low pH and started to increase as the pH solution increased. The optimum pH values for Ni²⁺ and Cd²⁺ biosorption were 6 and 5, at which the Ni²⁺ and Cd²⁺ biosorption values were 12.50 ± 0.68 and 21.97 ± 0.64 mg∙g⁻¹, respectively. This is in agreement with the character of metal cations biosorption. Successful biosorption of base metal cations usually takes place in the range of pH 3 - 7 and is extremely pH dependent [24] . An increase or decrease in the pH from these optimum pH values resulted in a reduction in the biosorption of these metal ions. At low pH values, protons in solution compete effectively with metals in binding to functional groups. pH effect may be further explained in relation to the competition effect between the hydronium ions (H₃O⁺) and metal ions [25] . As initial pH increases, the active sites are being deprotonated and strengthened the charge attraction, thus leading to significant increase in Ni²⁺ and Cd²⁺ biosorption. The decrease in biosorption of Ni²⁺ and Cd²⁺ ions above pH 6 is due to precipitation of both metals as insoluble hydroxides or hydrated oxides [22] [26] [27] .

The effect of contact time (15 to 120 min) on the equilibrium uptake of Ni²⁺ or Cd²⁺ ions onto Eup. ludwigii for an initial metal ion concentration of 50 mg∙L⁻¹ is shown in Figure 3. The biosorption capacity increased with increasing contact time and a large amount of metal ions was removed in the first 30 min. Equilibrium was reached in 45 and 75 min for Ni²⁺ and Cd²⁺, where the biosorption values reached 12.04 ± 0.71 and 22.23 ± 0.58 mg∙g⁻¹, respectively. After these equilibrium periods, either in Ni²⁺ or Cd²⁺ biosorption, the amount of adsorbed metal ions was not significantly changed with contact time. This rapid initial uptake was similar to the previous reports on the biosorption of these metals by different biosorbents. Akar and Tunali [28] observed that Cd²⁺ biosorption by Botrytis cinerea was fast and equilibrium was reached in 60 min. A contact time of 120 min was required to reach equilibrium in the biosorption of Cd²⁺ by Rhizopus cohnii [29] . Pahlavanzadeh et al. [1] noted that approximately 60% of Ni²⁺ ions were removed by the brown algae (Cystoseria indica, Nizmuddinia zanardini, Sargassum glaucescens, and Padina australis) in the first 20 min of contact, and equilibrium was reached

in a contact time of 120 min. This observed rapid biosorption of metals is among desirable parameters for successful deployment of the biosorbents for practical application [30] .

The initial metal ion concentration remarkably influenced the equilibrium metal uptake and biosorption yield. The effect of initial metal (Ni²⁺ and Cd²⁺) ion concentration was investigated in the range of 50 - 400 mg∙L⁻¹ under the determined optimum pH values and contact time. From the results presented in Figure 4, it was noted

that initial concentration increased the sorption of Ni²⁺ and Cd²⁺ ions, and then reached a saturation values at about 200 mg∙L⁻¹ for Ni²⁺ and 300 mg∙L⁻¹ for Cd²⁺. At these concentrations, the biosorbed Ni²⁺ and Cd²⁺ reached 26.23 ± 0.61 and 53.37 ± 0.84 mg∙g⁻¹, respectively. Then the values did not significantly change with the initial metal ion concentration. This increase in uptake capacity of both metals with increasing initial metals concentration is due to higher availability of metal ions for the sorption. Moreover, higher initial concentration provides increased driving force to overcome mass transfer resistance between the biosorbent and biosorption medium [31] .

The effects of temperature on biosorption of Ni²⁺ and Cd²⁺ onto Eup. ludwigii were carried out by varying a series of temperature from 15˚C to 40˚C. From the results presented in Figure 5, it was shown that the uptake amount of both metal ions increased with increasing temperature. So, the biosorption capacity increased from 3.67 ± 0.23 to 12.89 ± 0.21 mg∙g⁻¹ for Ni²⁺ and from 9.44 ± 0.27 to 23.72 ± 0.45 mg∙g⁻¹ for Cd²⁺ as the temperature increased from 15 to 40˚C. Similar results were reported on biosorption of Cd²⁺ onto Pycnoporus sanguineus [32] , Ni²⁺ onto some brown algae [1] , and Pb²⁺ onto Candida albicans [33] . The increase in the biosorption capacity with the increase in temperature indicates that the biosorption of Ni²⁺ and Cd²⁺ onto Eup. ludwigii was controlled by an endothermic process. This effect may be due to the fact that at higher temperatures an increase in active sites occurs due to bond rupture [1] . Opposite result was reported by Anayurt et al. [34] in the study on the biosorption of Pb²⁺ and Cd²⁺ by the fungus Lactarius scrobiculatus. They found that biosorption decreased from 98% to 90% for Pb²⁺ and from 95% to 88% for Cd²⁺ as temperature was increased from 20˚C to 50˚C.

To get the optimal biomass dose in Ni²⁺ and Cd²⁺ biosorption, different amounts (0.5 - 5 g∙L⁻¹) of fungal biomass were used. As can be seen from Figure 6, with the dose of biomass increasing, the metal uptake of Ni²⁺ and Cd²⁺ ions per unit mass of biomass (mg∙g⁻¹) was decreased. The biosorbed metal decreased from 38.22 ± 0.85 to 6.87 ± 0.21 mg∙g⁻¹ for Ni²⁺ and from 45.62 ± 0.78 to 9.83 ± 0.35 mg∙g⁻¹ for Cd²⁺ due to the increase in the fungal biomass from 0.5 to 5 g∙L⁻¹, respectively. This is expected because as the dose of biomass increased, there was increase in the available exchangeable sites for Ni²⁺ and Cd²⁺ ions. The maximum biosorption efficiencies reached 68.66% and 98.32% for Ni²⁺ and Cd²⁺, respectively, at biomass weight 5 g∙L⁻¹. Reduction in biomass dose in the biosorption medium at a given metal concentration enhanced the metal/biosorbent ratio and thus increased the metal uptake per unit weight of biosorbent as long as the later is not saturated [34] [35] .

In order to determine the optimal shaking rate, the biosorption of Ni²⁺ and Cd²⁺ ions by Eup. ludwigii was evaluated by varying the shaking rate of the biosorption media from 0 (without shaking) to 200. Figure 7 illustrates the effect of shaking rate (rpm) versus the amount of metal biosorbed (mg∙g⁻¹) of Ni²⁺ and Cd²⁺ ions. Control units at 0 rpm (no agitation) exhibited very low Ni²⁺ and Cd²⁺ uptake. At this shaking rate, the biosorbed Ni²⁺ and Cd²⁺ reached 2.45 ± 0.12 and 7.97 ± 0.24 mg∙g⁻¹, respectively. As shaking rate increased, the biosorption of Ni²⁺ and Cd²⁺ ions increased, and optimal values of biosorption were obtained for shaking rate of 150 and 100 rpm for Ni²⁺ and Cd²⁺ ions, respectively, and the biosorbed Ni²⁺ and Cd²⁺ were 13.34 ± 0.31 and 23.86 ± 0.64 mg∙g⁻¹, at the same order. Nearly, similar results were observed by Tun-Guo et al. [36] in the biosorption of Cd²⁺ by Aspergillus niger. They found that the optimal shaking rate was 120 rpm. The optimal values of Pb²⁺ and Co²⁺ biosorption capacity by Rhizopus oryzae were obtained at shaking rate of 150 rpm [9] . Opposite result was reported by Selatnia et al. [37] for biosorption of Ni²⁺ by a bacterial dead Streptomyces rimosus biomass. They found that the optimum shaking rate for biosorption of this metal is 250 rpm. The lower metal uptake of both metals at higher shaking rates is attributed to non-homogeneity of the biosorption mixtures caused by vortex phenomenon [38] , making the biosorption of Ni²⁺ and Cd²⁺ ions difficult.

The reusability and metal recovery efficiency of the biosorbent is likely to be a key factor in accessing the potential of the biosorbent for commercial application. As they are common elutants used by many researchers, 0.1 M HCl, 0.1 M CaCl₂, Na₂CO₃, EDTA, and ddH₂O were used to identify a suitable elutant agent. Table 1 shows the percentage of Ni²⁺ and Cd²⁺ ions released from fungal biomass after treatment with different elutants. From the results, it is evident that the desorption efficiencies of Ni²⁺ and Cd²⁺ ions were more than 96% after eluting with 0.1 M HCl, and so that HCl is the most efficient elutant to be used in the biosorption-desorption cyclic studies. HCl has been found to be an effective elutant for desorption of Ni²⁺ from Penicillium chrysogenum [22] and baker’s yeast [39] , and Pb²⁺ and Cd²⁺ from macro-fungus Lactarius scrobiculatus [34] . On the other hand, elution of Ni²⁺ and Cd²⁺ ions by ddH₂O exhibited negligible desorption capability and the desorption efficiency values do not exceed 3% for both metals, indicating a strong affinity of the fungal biomass towards Ni²⁺ and Cd²⁺ ions. After desorption using the most efficient elutant (0.1 M HCl), the biomass was washed with ddH₂O, and reused for another cycle. From the results illustrated in Figure 8, it is evident that there was a gradual de-

crease of Ni²⁺ and Cd²⁺ biosorption on Eup. ludwigii biomass with an increase the number of desorption cycles. After a sequence of five cycles, it was observed that the biosorption capacity of Eup. ludwigii biomass has been reduced to 75.2% and 80.8% for Ni²⁺ and Cd²⁺, respectively. The results indicate that Eup. ludwigii has good potential to adsorb these metal ions repeatedly from aqueous solution.

Biosorption isotherm provides a relationship between the concentration of metal in solution and the amount of metal on biosorbent when both the phases are at equilibrium. Modeling of Ni²⁺ and Cd²⁺ biosorption on the fungal biomass was realized by applying Langmuir and Freundlich adsorption isotherms. The linearized forms of the Langmuir isotherm model (Equation (5)) and Freundlich isotherm model (Equation (7)) were used to analyze and fit the data to these models. The Langmuir constants (q_max and b) with correlation coefficients (R²) were calculated from the plots in Figure 9 for biosorption of Ni²⁺ and Cd²⁺on the fungal biomass and the results are presented in Table 2. Also, Freundlich constants (K_f and n) with correlation coefficients (R²) were calculated from the plots in Figure 10, and presented in Table 2. The fit of experimental data to these models was evaluated by the correlation coefficients (R²). From the final results and based on the values of correlation coefficients (R²), Langmuir and Freundlich models best described the experimental data for biosorption of Ni²⁺ and Cd²⁺ at different temperatures, but biosorption of Cd²⁺ is more described by Freundlich model. In view of the Langmuir constant (q_max) values, the q_max values of Ni²⁺ and Cd²⁺ are close to the experimental q_max values at 20 and 30˚C, respectively. The favorable biosorption is indicating by higher than 1 value of Freundlich sorption constant n for fungal biomass. The values of n obtained greater than one for Ni²⁺ and Cd²⁺ indicated that physical and multilayer adsorption takes place for both metal ions. The small (K_f) values for Ni²⁺ indicate a lower extent biosorption, while more biosorption was observed for Cd²⁺ ions because of its larger (K_f) values. Generally, the higher values of Freundlich constants (K_f and n) and the lower value of Langmuir constant (b) indicating the higher affinity of the biomass [40] [41] . Similar results were also reported for biosorption of Ni²⁺ by Trichoderma viride [42] , Cd²⁺ by Rhizopus cohnii [29] , and Pb²⁺, Cu²⁺, and Cd²⁺ by Phanerochaete chrysosporium [43] .