Biosorption of cadmium using the fungus Aspergillus niger

Biosorption of cadmium using the fungus Aspergillus niger

L.M.Barros Júnior; G.R.Macedo^*; M.M.L.Duarte; E.P.Silva; A.K.C.L.Lobato

Universidade Federal do Rio Grande do Norte, UFRN, Departamento de Engenharia Química, DEQ. Programa de Pós Graduação em Engenharia Química (PPGEQ), Phone: [phone], Fax: [phone], Campus Universitário, 59072-970, Natal - RN, Brazil, E-mail: [e-mail]

ABSTRACT

Sorption experiments using the Aspergillus niger fungus for cadmium removal were carried out to study the factors influencing and optimizing the biosorption of this metal. The effects of pH, time, biomass concentration, and initial concentration of the heavy metal on the rate of metallic biosorption were examined. An experimental design was also used to determine the values of the under study variables that provided the greatest biosorption efficiency. A technique for biomass recovery was also developed with the objective of determining the capacity of the regenerated biomass to biosorb the metals in solution. This research proved that with a pH of 4.75, a biomass concentration of 0.7 g/L, and a heavy metal concentration varying between 5 and 10 mg/L a biosorption process of biosorption with Aspergillus niger could be successfully used for heavy metal removal from oil field water in the oil industry.

Keywords: Aspergillus niger, biosorption, experimental planning.

INTRODUCTION

Oil field waters, which are complex compounds of organic and inorganic matter found dissolved and in suspension, are one of several effluents generated by the oil industry. Some of the components of these effluents are heavy metals, often present at concentrations exceeding the limits allowed by the current legislation, thereby representing an environmental hazard (Braile and Cavalcanti, 1979).

Heavy metal ions from wastewater are commonly removed by processes of chemical precipitation, ion exchange and reverse osmosis. These methods have several disadvantages, such as unpredictable metal ion removal, high reagent requirements, and generation of toxic sludge which is often difficult to dewater and also requires extreme caution in its disposal (Kapoor et al; 1999).

Bacteria, fungi and seaweeds can be used to remove heavy metals and radioactive compounds from aqueous solutions (Brierley, 1990; Volesky, 1994). The uptake of heavy metals and radioactive compounds as a result of physicochemical interactions of metal ions with the cellular compounds of biological species is referred to as biosorption (Kapoor and Viraraghavan, 1998). Uptake of heavy metal ions by fungal microorganisms may offer an alternative method for their removal from wastewater. Fungi are used in a variety of industrial fermentation processes which could serve as an economical and constant source of supply of biomass for the removal of metal ions. Fungi can also be easily grown in substantial amounts using unsophisticated fermentation techniques and inexpensive growth media (Kapoor et al., 1999). Therefore, biosorption carried out by fungi could serve as an economical means of treating effluents charged with toxic metallic ions (Kapoor and Viraraghavan, 1995; Volesky and Holan, 1995)

The aim of this paper is to study biotechnological alternatives based on biosorption in order to study the effects of pH, heavy metal concentration, biomass concentration, and time on biosorption of cadmium in oil field waters. This method represents a biotechnological innovation as well as an excellent tool for use in the possible final states of oil field waters, such as in the water that surfaces, is reinjected and used for irrigation and domestic water supply after a simple treatment.

METHODS

Physicochemical Characterization of Oil Field Waters

Samples of oil water were collected from the effluent treatment station at Guamaré, a city in the state of Rio Grande do Norte, Brazil. The rate of concentration of heavy metal in the oil field water was determined by atomic absorption. An atomic absorption spectrophotometer (AAS) was used for this purpose.

Biomass Production

A fungal biomass was cultivated in liquid phase using a rotating incubator. The suspension of spores was transferred to 250 ml Erlenmeyer flasks filled with 100 ml of a culture medium composed of the following (g/l): bacto dextrose, 20; bacto peptone, 10; NaC1, 0.2; CaC1_2. 2H₂O, 0.1; KCl, 0.1; K₂HPO₄, 0.5; NaHCO₃, 0.05; MgSO₄, 0.25 and Fe(SO₄). 7H₂O, 0.005. The liquid phase pH was adjusted to 5 by the use of 1 N HCl. Once inoculated, the flasks were shaken on a rotary shaker at 125 rpm for five days at 25^oC. The biomass produced was collected by vacuum filtration and boiled in 0.5 N NaOH solution for 15 min (Kapoor et al., 1999). It was then washed with generous amounts of deionized water as long as the pH of the washing solution was in the near-neutral range (7.0 – 7.2). After washing, the biomass was dried at 60^oC for a period of 16 h and powdered to be used in biosorption tests in series.

Biosorption Experiments

With the objective of achieving an understanding of the process of biosorption to establish better conditions for this process and to provide data for the biotechnological treatments based on biosorption, sorption experiments were carried out using stock solutions of metallic cadmium (99.99%, MERCK) in order to study the effects of pH, time, biomass concentration, and initial concentration of the heavy metal in the process of removal of metallic cadmium at a constant temperature. The heavy metal concentrations were determined using an atomic absorption spectrophotometer. All the biosorption experiments were conducted in 250 mL Erlenmeyer flasks on a rotary shaker (at 125 rpm) at room temperature (25ºC) (Kapoor and Viraraghavan, 1998). All the experiments were conducted in duplicate and mean values were used in the analyzing the data.

The amount of metallic ion biosorbed by g of the biomass (q) and the efficiency of biosorption (E) were calculated using equations 1 and 2, respectively:

where C_i is the initial concentration of the metallic ion (mg L^-1), C_f is the final concentration of the metallic ion (mg L^-1), m is the mass of the biosorbent in the reaction mixture (g), and V is the volume of the reaction mixture (L).

With a view to determining the influence of pH, cadmium concentration, and biomass concentration (independent variables) on the efficiency of biosorption (dependent variable ), an experimental design was used.

Experimental Design

The biosorption process studied was modeled based on an experimental design. In this study the Modreg and Statistic programs for Windows, Release 5.5, were used. The first eight experiments were carried out using a combination (upper and lower levels) of independent variables; three experiments at the central point and six experiments at a distance of 1.2 from the central point (at codified variables). The efficiency of biosorption (E) for each experiment was defined by equation 2. The time used for these experiments was 6 h. Table 1 contains the experimental matrix with the factors in codified form. A total of 17 experiments were carried out and of these three were repeated at the central point (experiments 9, 10 and 11, Table 1).

Biomass Recovery

After the biosorption tests the biomass was washed with deionized water for 15 minutes and left in 25 mL of 0.05 N nitric acid for one hour at 25^oC in a rotating shaker. The biomass was separated from the solution by vacuum filtration and washed with deionized water until the pH of the filtrate reached 5 – 5.4. Then it was stirred into a solution containing 0.001 M Ca⁺⁺, Mg⁺⁺, and K⁺ for 30 minutes at 125 rpm in a rotating shaker. The recovered biomass was dried in an oven at 60^oC and the capacity to biosorb metal was determined (Kappor et al., 1999). The biosorption – desorption cycle of cadmium metal-biomass recovery was repeated four times in order to determine the biosorption capacity of the recovered biomass.

RESULTS AND DISCUSSIONS

Physicochemical Characterization of the Oil Field Water

The results of the physicochemical analysis of the main polluting heavy metals found in the oil field water generated in the oil fields of the state of Rio Grande do Norte, Brazil are shown in Table 2.

From the results obtained, it was observed that the concentrations of cadmium and soluble iron were higher than the levels permitted by the environmental legislation (Article 4 of CONAMA 20). For the biosorption tests in series, a model cadmium solution with concentrations higher than the one used in the oil field water, since the range of detection for the atomic absorption spectrophotometer (0.2 – 3 mg/L of cadmium) is higher than that verified in Table 2.

Effect of pH on Biosorption

The influence of hydrogen ion concentration on the biosorption of cadmium was studied by varying the pH of the metal solution in the biosorption experiments. The effect of pH on the biosorption of cadmium by the Aspegillus niger biomass can be seen in Figure 1.

There was little or no biosorption of metallic cadmium when the pH was lower than 3. Removal capacity increased rapidly when pH increased from 3.0 to 4.0, and then starting at pH 4.0 the increase in biosorption capacity showed to a less pronounced rate. This could be explained by the increase in density of the negative charge on the cell surface, causing proton removal on the cell bonding sites, thereby increasing its biosorption capacity. For Aspergillus niger, the optimum biosorption (mg of Cd / g of biomass) and lowest equilibrium concentration (mg of Cd / L) were obtained for a pH ranging between 4.0 and 5.5. This range was selected for the experimental design.

Analysis of the Kinetics of Biosorption of Cadmium by Aspegillus Niger

Studies on the kinetics of the process of cadmium removal by Aspegillus niger were carried out with the purpose of observing the evolution of the process up to when the system reached equilibrium. The biosorption of cadmium in relation to time at different pH values is shown in Figure 2.

The concentration of cadmium decreased with increasing contact time and reached equilibrium (constant biosorption) after 6 h at pH 4.0, 5.0, and 5.5. The plots showed that the kinetics of biosorption of cadmium consisted of two phases: an initial rapid phase where biosorption was fast and significantly contributed to equilibrium uptake and a slower second phase where the contribution to total metal biosorption was not significant. It was also verified that in one hour the biomass reached 91% of its saturation capacity at pH = 5.0 and from the 6^th to the 10^th hours, there was no significant removal of cadmium. The kinetics of heavy metal adsorption can be modeled by the pseudo-second-order Lagergren equation (Kapoor et al., 1999), shown below as Equation (3):

where K^´ is the second-order rate constant for adsorption (g mg^-1 h^-1), q_e the amount of metal ion adsorbed at equilibrium (mg g^-1), and q_t the amount of metal ion adsorbed (mg g^-1) at any given time t (h). Figure 3 shows the experimental data (observed values) and the pseudo-second-order Lagergren model (predicted values) for biosorption of cadmium at different pH values.

The kinetic data for cadmium biosorption, which was fitted to a pseudo-second-order reaction-rate model, had a high correlation coefficient (0.92 – 0.94) with a confidence level of 95%.

The Influence of Biomass Concentration

Experiments on the influence of biomass concentration on the process of cadmium removal by Aspegillus niger were carried out with the purpose of observing the effect of this parameter on the rate of metallic biosorption. The results obtained are shown in Figure 4.

Biomass concentration is an important parameter which affects efficiency and sorption capacity. Whenever there was an increase in biomass concentration, there was an increase in efficiency and a decrease in sorption capacity. For an initial concentration of the metal used (5.3 mg/L), no change was observed in the biosorption efficiency for 0.7 g/L of biomass concentration. The sorption efficiency curve was used as a parameter for the statistical analysis, and 0.4 and 0.7 g/L were defined as the lower and upper levels of biomass concentration to be used in the experimental design.

The Influence of Concentration of Synthetic Cadmium Solution

Experiments on the influence of concentration of synthetic cadmium solution on the removal of cadmium by Aspegillus niger at fixed values of pH and biomass concentration (5.5 and 0.7 g/L) were carried out with the purpose of observing the effect of this parameter on the rate of metallic biosorption. The results obtained are shown in Figure 5.

The results showed that increasing the initial concentration of the metal increased the sorption capacity increased up to 30 mg/L and then decreased it from this point on. Sorption efficiency decreased when the initial concentration of cadmium increased. Therefore, in order to achieve greater efficiency in the removal of the heavy metals, it is necessary to maintain the effluent at a low metal concentration (< 5), which enables removal of heavy metals from the oil field water in the petroleum industry (Table 2). Due to the limitations of the detection ranges of the atomic absorption spectrophotometer, 5 and 10 mg/L were selected as the lower and upper levels of the initial concentrations of cadmium. The results of the experimental range for each independent variable are shown in Table 3.

Biosorption Isotherm Analysis

The biosorption equilibrium of heavy metals was modeled using adsorption-type isotherms. The Langmuir and Freundlich models were used to describe biosorption equilibrium (Kappor et al., 1999). The form of the Langmuir model is

and the form of the Freundlich model is

where q_e is the sorption capacity at equilibrium (mg g^-1), q_m is the maximum sorption capacity (mg g^-1), C_e is the equilibrium concentration of metal ion (mg L^-1), b the Langmuir model constant (L/mg), and K and n the Freundlich model constants (dimensionless).

The batch biosorption data were fitted to both models by nonlinear regression analysis using the software package Statistic software package (Release 5.5) for Windows. The Langmuir and Freundlich equations were modeled using the Quasi-Newton algorithm. Table 4 shows the model constants along with correlation coefficients for biosorption of cadmium by Aspergillus niger.

The Langmuir model parameters obtained were statistically significant at all pH values studied at a confidence level of 95%. The Freundlich parameters estimated were not significant at all pH values studied at a confidence level of 95%.

Figure 6 shows the Langmuir plot for biosorption of cadmium by Aspergillus niger at different pH values. The biomass concentration was 0.7 g/L and the time was 6 h.

Experimental Design

With the aim of determining the pH values, initial cadmium concentration, and biomass concentration in order to achieve greater efficiency in the biosorption of cadmium by the Aspergillus niger biomass, an experimental design was used within the experimental range defined in Table 3.

The software Statistic for Windows, Version 5.5 was used to analyze the experimental design. Figure 7 represents Pareto's graph with a confidence level of 95% for estimating the main linear and quadratic effects and the effects of the second order for absolute values. The magnitude of each effect is represented by bars, and a dashed line corresponding to the value of p = 0.05 indicates how big the effect should be in order to be of statistical significance.

Analysis of Figure 7 shows that the most important points for determining the efficiency of biosorption of cadmium by Aspergillus niger are the main linear effects of biomass concentration C(L), pH A(L), and initial concentration of cadmium B(L); the interaction effects between initial concentration of cadmium and biomass concentration, B(L) by C(L), and pH and biomass concentration, A(L) by C(L); and the quadratic effects of the pH A(Q) and biomass concentration C(Q).

The linear model with a confidence level of 95% obtained for the response function Y is represented by equation (6):

where the values of variables A, B, and C are coded.

The linear model had a regression coefficient (R²) of 0.81828 and for a confidence level of 95%, the value of F calculated for the model was 3.09 times higher than that in the table (Barros Neto et al., 1996), F_model = 9.90 and F_(5/11)table = 3.20.

The quadratic model obtained for the response function Y is represented by equation (7):

where the values of variables A, B, C, and D are coded.

Variance and regression analysis demonstrated the statistical significance of the model, the regression coefficient was higher than the linear model, R² = 0.9339, and for a significance level of 95% the value of F calculated according to the model was 5.53 times higher than that in the table (Barros Neto et al., 1996), F_model = 18.18 and F_{(7/9) table} = 3.29, justifying the use of the quadratic model for statistical analysis.

Equation 8 is used in transforming coded values into real values of the variables studied in the experimental design.

where x values are coded and y values are real.

The three experiments carried out at the in central point (experiments 9, 10, and 11, Table 1) have a low regression coefficient (0.00038), indicating that the residue due to pure error didn't interfere with the regression analysis of the obtained data.

The studies on the main quadratic and linear effects and their interactions on the efficiency of biosorption of cadmium by Aspegillus niger (answer Y) could be carried out using the isoresponse curves. The greatest efficiency for biosorption of cadmium could be identified through inspection of these curves.

The isoresponse curve (Figure 8) represents the efficiency of biosorption of cadmium by Aspergillus niger in relation to the main linear and quadratic effects and their interactions.

For the biosorption of cadmium at pH 4.75 with a fixed quantity of biomass of 0.050 g for 75 mL of cadmium solution for any value of initial cadmium concentration, the efficiency of the biosorption of cadmium by Aspergillus niger was around 96.98% (Figure 8).

The final results showed that the process of biosorption of cadmium by Aspergillus niger could be carried out efficiently using the following operational conditions: a pH of 4.75, an initial cadmium ion concentration varying between 5 and 10 mg/L, and a biomass concentration of 0.70 g/L.

Desorption of the Biosorbed Cadmium and the Recovery of the Biomass.

The use of fungal biomass with a potential biosorbent depends not only on its biosorption capacity but also on how it can be reused. Biomass was used for four cycles of biosorption – desorption of biosorbed metal cadmium to study the changes in biosorption capacity and its subsequent reuse. In Figure 9 it can be observed that the biomass lost part of its biosorption capacity during the last cycles. The biomass lost around 86% of its biosorption capacity after the fourth cycle, and it was necessary to study the economic viability of the application of Aspergillus niger biomass in industrial processes.

CONCLUSIONS

It was concluded that pH was an important factor in the process of cadmium sorption by the Aspergillus niger biomass, showing an increase in biosorption capacity with an increase in pH. The kinetics of biosorption was rapid, showing that the initial 84% of biosorption capacity was achieved in the first 5 min of contact with a biomass concentration of 0.70 g/l, a cadmium concentration of 10 mg/L and a pH of 5.0. An increase in efficiency and a decrease in sorption capacity occurred with an increase in biomass concentration. An increase in biosorption capacity and a decrease in biosorption efficiency result in an increase in initial concentration of cadmium.

The efficiency of biosorption could be represented as a function of operational variables such as pH, initial concentration of the synthetic cadmium solution, and biomass concentration, by the following quadratic model:

Y = 91.37 + 4.55A – 3.33B + 7.74C –

– 4.97A² – 1.46C² – 2.15AC + 2.66BC

The following experimental conditions were selected to guarantee more efficiency in the process of biosorption of cadmium by Aspergillus niger: a pH of 4.75, an initial cadmium concentration of 5 - 10 mg/L and a biomass concentration of 0.70 g/L.

In relation to recovery, it was observed that Aspergillus niger lost 76.4% of its biosorption capacity after the first cycle and was able to maintain the rest of its capacity for three more cycles.

This work demonstrated that the Aspergillus niger biomass has considerable potential in removing heavy metals from oil field waters of the petroleum industry.

NOMENCLATURE

ACKNOWLEDGEMENT

The authors would like to thank ANP (National Petroleum Agency - Brazil) for their financial support in carrying out this work.

Received: March 13, 2002

Accepted: April 4, 2003

• Barros Neto, B., Scarmínio, I.S., and Bruns, R.E., Planejamento e Otimização de Experimentos. 1st ed. Campinas: Editora da Unicamp, 299p., 1996.
• Braile, P.M., Cavalcanti, J.E.W.A., Manual de Tratamentos de Águas Residuárias Industriais. 18th ed. São Paulo: CETESB, 1979.
• Brierley, C.L., Bioremediation of Metal-contaminated Surface and Groundwaters. Geomicrobiology Journal, 8, 201, 1990.
• Kapoor, A. and Viraraghavan, T., Biosorption of Heavy Metals on Aspergillus niger: Effect of Pretreatment. Bioresource Technology, 63, p. 109, 1998.
• Kapoor, A. and Viraraghavan, T., Fungal Biosorption An Alternative Treatment Option for Heavy Metal Bearing Wastewater: A Review. Bioresource Technology, 53, 195, 1995.
• Kappor, A., Viraraghavan, T. and Cullimore, D.R., Removal of Heavy Metals Using the Fungus Aspergillus niger. Bioresource Technology, 70, 95, 1999.
• Volesky, B., Advances in Biosorption of Metals: Selection of Biomass Types. FEMS Microbiology Review, 14, 291, 1994.
• Volesky, B. and Holan, Z.R., Biosorption of Heavy Metals. Biotechnology Progress, 11, 250, 1995.

*

To whom correspondence should be adress

Publication Dates

• Publication in this collection
  01 Sept 2003
• Date of issue
  Sept 2003

History

• Accepted
  04 Apr 2003
• Received
  13 Mar 2002