Microorganism
Aspergillus tamarii NRC 3 was isolated from Egyptian soil and identified by molecular identification using 18S rRNA (Saad et al. 2016).
Maintenance of the isolated fungus
Potato dextrose medium (PDA) was used for maintenance of the isolated fungus. The inoculated slants were incubated for 7 days at 28 °C. The cultures were maintained at 4 °C and subcultured every 2 weeks.
Culture method
The spore solution was inoculated into Petri dishes each containing 20-ml PDA medium, pH 5. The cultures were incubated at 28 °C for 6 days.
Growth conditions
One disk (4-mm diameter, equal 2 × 107 spore) was inoculated into a sterilized mannanase production broth medium: modified Czapek’s Dox medium contains (g/l) locust bean gum 10.0; NaNO3, 2.0; K2HPO4, 1.0; MgSO4.7H2O, 0.5; KCl, 0.5; and traces of FeSO4.7H2O (pH 5). The flasks were incubated statically for 6 days at 30 °C. At the end of incubation period, the fungal biomass was separated from the culture by filtration, washed several times with distilled water, and dried between two filter papers to obtain wet biosorbent and the clear supernatant was considered as the crude β-mannanase enzyme (Saad et al. 2016).
Preparation of heavy metal solutions
The 1000-ppm stock solutions of Cu2+, Pb2+, Co+ 2, Ni2+, Fe+ 3,or Cr3+ were made in double distilled water using CuCl2, PbCl2, CoCl2, NiCl2, FeCl3, or CrCl3 (Merck or BDH). The 10 to 120 ppm solution of Cu2+ and 25 ppm from each tested metal were prepared from 1000 ppm stock solution by dilution with double distilled water.
Analytical methods
Amount of metal taken up by the biomass was calculated by difference in initial and final concentration in the solution. The residual metal ions were determined by atomic absorption spectrophotometer FS240Agilen.
Heavy metal ions uptake by Aspergillus tamarii NRC 3 biomass
General procedure (Saad 2015)
The pre-cultured fungal biomass (5 g wet weight = 0.44 g dry weight) live or pretreated biomass (dead biomass) was added to 50 ml of metal(s) solution in 250 ml flasks. Duplicates of these flasks were incubated in a shaking incubator at 150 rpm for interval time at 30 °C. A 50-ml metal(s) solution without biomass was incubated in the same manner and stored as control. By the end of the experiment, the content of each flask was filtered through filter papers (Whatman No.1). The biomass was removed from the filter paper, and the supernatant was analyzed for the determination of residual metal(s).
Kinetic and mechanism of Cu2+uptake by Aspergillus tamarii NRC 3 biomass
Factors that influence copper(II) uptake by fungal biomass were investigated as follows.
Equilibrium experiments
In order to determine the maximum time of equilibration for the maximum sorption of Cu2+ by the biomass, several equilibrium experiments were conducted at different times ranging from 5 min to 120 min.
Effect of Cu2+ concentration
The dependence of Cu2+ uptake on initial Cu2+ concentration (10–120 ppm) was performed at 1 h period.
Effect of biomass concentration
The effect of different biomass concentrations (1–5 g wet weight) on the Cu2+ uptake was studied.
Effect of pH
The effect of pH on Cu2+uptake was performed with the range of 3–9 by adjusting the metal solution to the desired pH with either 0.1 N NaOH or 0.1 N HCl before adding the biomass.
Effect of temperature
The effect of different temperatures (10, 20, 30, 40, and 50 °C) on the uptake of Cu2+ during 1 h period was investigated.
Effect of mixed metal ions on Cu2+ uptake
The effect of a mixture of competing metal ions, i.e., Pb2+, Co+ 2, Ni2+, Fe+ 3, and Cr3+ in a concentration of 25 ppm of each metal in addition to Cu2+ (100 ppm) on Cu2+ uptake, was studied. A 50-ml Cu2+solution in a concentration of 100 ppm with fungal biomass was incubated in the same manner as the control.
Effect of pretreatment of fungal biomass on Cu2+ uptake (Saad 2015)
Fungal biomass (each 5 g, wet weight = 0.44 g dry weight) was treated prior to contact with Cu2+solution (100 ppm of Cu2+, pH 5) as follows:
-
1.
Boiling with distilled water for 10 min
-
2.
Soaking in 5% KOH solution for 10 min, separated by filtration and washed with 1.0 N HCl then thoroughly washed several times with distilled water until neutral
-
3.
Soaking in 1.85 × 10−5 mM of sodium azide solution for 30 min, separated by filtration and washed several times with distilled water
Appropriate controls for the treatment were prepared and treated simultaneously as the experiments.
Sorption and desorption of mixed metal ions
Sorption of mixed metal ions, i.e., Cu2+, Pb2+, Co+2, Ni2+, Fe+3, and Cr3+ was carried out as outlined in the general procedure. After initial metal ions uptake, the fungal biomass was separated by filtration and repeatedly washed with distilled water. The metal ions content in the supernatant was determined. The metal ions sorbed by the biomass were taken as 100% for the subsequent uptake cycle.
The biomass obtained from the initial biosorption (described above) was subjected to desorbing agents (0.1 N HCl, 0.1 N NaHCO3, or 0.1 N Na2CO3).
The biomass was suspended in 50 ml of desorbing agent for 1 h. Another 50 ml of desorbing solution without the biomass served as a control. The biomass was separated by filtration. The metal ions content in the supernatant was determined. The elution efficiency of the desorbing agent was calculated as follows:
Elution efficiency % = amount of desorbed metal ions/amount of biomass sorbed metal ions × 100
Metal ions uptake by the regenerated biomass
The biomass obtained after the desorption treatment was washed repeatedly with distilled water and re-suspended in 50 ml of metal ions solution for 1 h. Another 50 ml portion of metal ions solution was incubated separately and served as a control. By the end of incubation period, the biomass was separated by filtration and metal ions were determined in supernatant.
Reloading capacity = amount of sorbed metal ions in the second cycle/amount of sorbed metal ions in the first cycle
The experimental procedures above were carried out with each of the desorbing agent in duplicate along with the appropriate controls.
Removal of metal ions from industrial effluent
An industrial effluent was obtained from the Egyptian Company for leather tanning (El-Basateen, Cairo).
Effluent treatment
The removal of metal ions from the industrial effluent was carried out as outlines in the general procedure.
Statistical analysis
Data are expressed as the mean ± S.E. of three independent culture preparations performed in triplicate. Statistical analysis was achieved using prism software-programmed one-way ANOVA.