Biosorption of Heavy Metals from Aqueous Solutions

St.Mihova, T.Godjevargova

University ”Prof. Dr. A. Zlatarov”, Bourgas 8010, Prof Y.Yakimov Str.1

Received 18.07.01; Cited 19.08.01

 Abstract

Removal of heavy metals from waste waters is a major ecological problem. In recent years it has been solved by using microorganisms as means to extract heavy metal ions. In this connection, the kinetics of growth and sorption of Cu²⁺ by four types of cells was studied: Aspergillus niger, Phanerochaete chrysosporium, Saccharomycopsis lypolytica and Saccharomyces cerevisae. Better sorption properties were observed to have the yeast S.cerevisiae and the fungi Ph.chrysosporium. The effect of Cu²⁺ concentration on sorption and growth of these strains were studied in details. It was found that the biomass accumulated decreased with the increase of Cu²⁺ concentration. Cuprous ions with concentration of 50 mg/l exert a weak inhibiting effect and with concentrations higher than 250 mg/l the period of cells adaptation was longer  and their growth - slower.

The sorption of Cu²⁺ by the two strains studied was proved to be a fast process. Up to 75% of the Cu²⁺ content were absorbed during the first 6 hours.

The adsorption capacities of the two sorbents were calculated. Higher adsorption capacity was found to possess S.cerevisiae (3.5 mg/g) than Ph.chrysosporium (2.5 mg/g).

The sorption rate of the fungi was higher. The rate constants were compared using the Lagergren plot drawn for both types of cells studied. Higher values of k showed S.cerevisiae.

The effect of the type of metal on the sorption process was studied and the following order of sorption was observed for both types of cells: Pb²⁺>Cu²⁺>Ni²⁺.

The results obtained exhibit the potential of S.cerevisiae and Ph.chrysosporium to be used for decontamination of waste waters containing heavy metal ions.

Kew wards: biosorption, heavy metals, free cells, kinetics

 Introduction

In recent years the process of accumulation of heavy metal ions by micro organisms was intensly studied. Microorganisms like bacteria, yeast and fungi [1], as well as algae [2] can accumulate large amounts of heavy metal ions.

Biosorption is considered to be a fast physical or chemical process. The biosorption rate depends on the type of the process. According to literature, biosorption can be divided into two main proceses: adsorption of the ions on cell surface and bioaccumulation within the cell [3].

The bioaccumulation of heavy metals is closely connected with their toxicity, i.e. restraining microorganisms metabolism and growth [4,5]. Low concentrations of heavy metal ions are necessary for the vitality of all microbial cells and certain low concentrations of Cu and Zn even stimulate the growth and the activity of the metabolic processes [4,6]. At high heavy metals concentration the growth may be severely restrained which can be observed as a prolonged lag-phase [7]. The values of the critical and threshold concentrations depend on the type of the microorganism and the properties of the metal. The metal toxicity is affected also by the form in which they exist in the medium, the amount of cells in the medium and the stage of growth of the cell culture.

The aim of the present paper is to study the biosorption of heavy metal ions by two strains: S.cerevisiae and Ph.chrysosporium, as well as their effect on  biomass growth.

Materials and methods

Microorganisms

The strains Saccharomyces cerevisiae and Phanerochaete chrysospotium were isolated, determined and provided by the Institute of Microbiology of the Bulgarian Academy of Sciences, Sofia.

Both strains were maintained on beer agar.

Cultivation

The strain S.cerevisiae was cultivated in a nutrition medium containing b-D-glucose (10 g/l), malt extract (3 g/l), yeast extract (3 g/l), peptone (5 g/l).

The strain Ph.chrysosporium was cultivated in a medium containing 20 g/l glucose, 20 g/l malt extract and 1 g/l peptone.

In a test tube containing fresh culture on a beer agar, a biomass was washed out with physiological solution. Under sterile conditions, in an Erlenmeyer flask were placed 45 ml nutrition medium and 5 ml cell suspension, to obtain suspension containing cells with concentration 0.5 g/l dry weight. The cultivation was carried out on a shaker at 220 rpm and 30°C.

For the experiments on the effect of the heavy metal ions on both strains, they were added to the nutrition medium in different concentrations. They were added to the 24^thh culture of Phanerochaete chrysosporium and in the same beginning of cultivation of Saccharomyces cerevisiae. The following salts were prepared to obtain ions of heavy metals: CuSO₄x5H₂O, Ni(NO₃)₂x6H₂O, Pb(NO₃)₂ with concentrations of 10 g/l. The solution of metal salts were sterilized in an autoclave at 120°C for 30 min.

Determination of cell growth

The concentration of cells on the cultural medium was determined by measuring the optical density of the cell suspension at l=610 nm in a cuvette with standard thickness of 0.998 cm on a “Specol-11” apparatus (Carl Zeiss Jena, Germany).

The amount of dry biomass in the suspension was determined by samples taken at 2 h intervals from the cultural medium, which were then centrifuged at 4000 rpm for 20 min and the precipitate was dried at 85°C until constant weight.

Determination of the Cu²⁺ ion concentration

Samples of 2 ml were taken at 0, 1, 3, 6, 24 and 48 h from the cultural medium. The samples were centrifuged and the supernatant was subjected to atomic absorption analysis to detemine the residual concentration. The atomic absorption spectrophotometer AAC-1 (Carl Zeiss Jena, Germany) used was equipped with hollow-cathode lamp for ice and a slit burner for air-acetylene.

Kinetic study

The metal uptake (q) for the construction of sorption isotherms was determined as follows:

 where q is adsorption capacity (mg Cu²⁺/g of biomass), V is the volume of solution in the contact batch flask (ml), C_i- initial concentration of Cu²⁺ in the solution (mg/l), C_f- the final concentration of Cu²⁺ in the solution (mg/l), W- mass of cells (g).

Lagergren presents the adsorption kinetic of a first order rate expression:

where q_f- is the amount of Cu²⁺ adsorbed (mg/g) at equilibrium time, q_t is the amount of Cu²⁺ adsorbed at time t (h), K_ad - rate constant of adsorption (h^-1). Linear plot of log (q_f - q_t) vs t gives the constant K_ad.

Results and Discussion

The kinetics of growth of four types of strains was studied: Aspergillus niger, Phanerochaete chrysosporium, Saccharomycopsis lypolytica and Saccharomyces cerevisae in the presence of Cu²⁺ with concentration 50 mg/l (Fig.1a). The highest growth was observed for A.niger and Ph.chrysosporium. The growth of biomass in the initial period was higher for S.cerevisiae, but after 24 h it was found a little slower than that of A.niger and Ph.chrysosporium.

Fig.1a. Growth kinetics of four strains in the presence of Cu²⁺ (50 mg/l): n- Aspergillus niger; Δ- Saccharomyces cerevisiae; u- Saccharomycopsis lipolytica; 6-Phanerochaete chrysosporium

 At the same time, the sorption of Cu²⁺ with initial concentration 50 mg/l by these strains was studied (Fig.1b).The best sorption properties for Cu²⁺ ions showed S.cerevisiae and Ph.chrysosporium. Based on the results obtained, the further experiments were carried out with these two strains.

Fig 1b. Sorption kinetics of Cu²⁺ (50 mg/l) by different strains: n- Aspergillus niger; Δ- Saccharomyces cerevisiae; u- Saccharomycopsis lipolytica; 6-Phanerochaete chrysosporium

The effect of Cu²⁺ concentration of biomass growth of both strains was studied (Fig.2). Three Cu²⁺ concentrations were used: 50, 100 and 250 mg/l. These concentrations were selected following a preliminary experiment for the growth of strain S.cerevisiae at Cu²⁺ concentrations from 50 to 400 mg/l, which showed that at concentrations higher than 300 mg/l the biomass growth significantly decreased.

 a

 b

Fig.2 Growth kinetics of the two strains S.cerevisiae(a) and Ph. chrysosporium(b)

Fig.2a shows that stronger inhibiting effect exerted Cu²⁺ ions with initial concentration of 250 mg/l. At 50 mg/l Cu²⁺ ions this effect was found to be negligible. Similar dependence was observed with the cells of strain Ph.chrysosporium, with the only difference that Cu²⁺ concentrations of 250 mg/l had stronger toxic effect which almost stopped cells growth. As can be seen from Fig.2b, the development of strain Ph.chrysosporium had not reached stationary phase 48 h after the beginning of cultivation. For full characterization of this process, future studies should cover the biosorption of heavy metals to  later stages of the development of strain Ph.chrysosporium.

 a

 b

Fig. 3 Sorption kinetic of Cu²⁺ with different concentrations by free cells of S.cerevisiae(a) and Ph. chrysosporium(b)

Comparing the kinetics of Cu²⁺ biosorption by both strains, it can be seen that the differences were scarce (Fig.3). Both processes took place with high rate (70-75% Cu²⁺ ions were absorbed during the first 6 h). The sorption capacities (q) of both strains were calculated and are presented in Fig.4. The sorption capacities increased with the initial metal ions (Cu²⁺) concentration. The cells of strain S.cervisiae (Fig.4a), however, showed higher capacity than that of Ph.chrysosporium (Fig.4b).

a

b

 Fig 4. Adsorption capacities of free cells of strain S.cerevisiae(a) and Ph. chrysosporium(b)

For better characterization of the sorbents, the plot log(q_f-q_t) was drawn (Fig.5) for the period of the initial 6 hours of the experiments (because the major part of Cu²⁺ was absorbed in this period) for both strains.

 a

 b

Fig 5. Lagergren plot for sorption of Cu²⁺ with different concentrations on free cells of strain S.cerevisiae(a) and Ph. chrysosporium(b)

The rate constants K_ad for each biosorbent studied was calculated by the slope of the corresponding straight line and their values are presented in Table 1. The results obtained show that the rate constants of cells of strain Ph.chrysosporium were higher than these of S.cerevisiae.

Table 1. Values of biosorption constant Kad, h^-1

 a

 b

Fig. 6. Sorption kinetic of Pb²⁺,Ni²⁺ and Cu²⁺(50 mg/l) by free cells of strain S.cerevisiae(a) and Ph. chrysosporium(b)

The effect of the type of metal on the sorption process was studied. The sorption abilities of both strains to Pb²⁺ and Ni²⁺ ions were compared with the results for Cu²⁺ (Fig.6). Obviously, Pb²⁺ ions were sorbed better than the other heavy metals and the sorption reached up to 90% of the initial concentration. Nickel ions were least sorbed by both strains. It can be see also that the sorption curves of Ni²⁺ and Cu²⁺ were very close, especially with cells of fungi Ph.chrysosporium.

Table 2. Adsorption capacities of free cells for different metal ions at 48 h

The sorption capacities of the two strains to Cu²⁺, Pb²⁺ and Ni²⁺ were calculated (Table 2). The results showed that the metals studied can be arranged in the following order according to their sorption from both types of cells: Pb > Cu > Ni. The adsorption capacity of S.cerevisiae and Ph.chrysosporium for Pb²⁺ was found to be about 3 times higher than the results for the other two metals.

The results obtained prove the potential of the cells studied for purification of waste waters from heavy metal ions.

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