بررسی جذب زیستی یون‏های مس توسط کیتوزان قارچی: جایگزینی برای کیتوزان میگویی

Introduction

Chitosan is a non-toxic and biodegradable polysaccharide containing amino and hydroxyl groups which makes it a suitable adsorbent for hazardous heavy metal removal. Industrially, chitosan is produced through deacetylation of chitin, which is mainly obtained from shellfish waste (1), with hot concentrated sodium hydroxide (2 and 3). Seasonal problems and limited supply in some regions reduce the industrial production of chitin and chitosan. Moreover, chitosan quality and the processing expenses are affected using a strong base solution at high temperatures to convert chitin to chitosan. An alternative source of chitosan is the cell wall of zygomycetes fungi (4 and 5). Fast fungal growth on inexpensive substrates regardless of season or location, lower concentration of inorganic material in fungal biomass, and enzymatic deacetylation of chitin to chitosan in the cell wall of zygomycetes instead of the expensive chemical deacetylation step for chitosan production are among the benefits of zygomycetes fungi over shellfish wastes for production of chitosan. Therefore, production of chitosan using fungi may be industrially advantageous and environmentally acceptable (6 and 7).

Copper is a serious water pollutant present in sewage of industries such as metal cleaning, paper and paperboard mills, and fertilizer production (8 and 9). Although trace amount of copper is essential as a nutrient, this metal is severely toxic and harmful for plants and humans at high concentrations (10 and 11). Maximum permissible limit of copper ions in drinking water is 2 mg l^-1, since it accumulates in the liver at higher concentrations (12).

Several reports are available studying the biosorption of copper ion using chitosan obtained by deacetylation of chitin available in crustacean such as shrimp (13 and 14) . However, to the best of our knowledge, there is no comparative report on the biosorption of Cu²⁺ by chitosan produced from deacetylation of shrimp chitin (shrimp chitosan) and chitosan extracted from the cell wall of fungi (fungal chitosan), and the maximum adsorption capacity (q_max) of fungal chitosan and the effects of operating parameters on copper removal by fungal chitosan have not been studied comprehensively.

In the current work, shrimp and fungal chitosans were used to remove copper ions. Mucor indicus biomass was used since it contains adequate quantities of chitosan (15). M. indicus is widely used in fermentation of lignocellulosic hydrolysates and production of a number of valuable materials, e. g., fishmeal, fungal extracts, and fatty acids (16). The high-quality fungal chitosan was separated and purified based on a recently developed method (17) using sulphuric acid, instead of conventional methods and using acetic acid or hydrochloric acid, which produce chitosan with a higher purity and yield. A comparative study on adsorption of copper ions by shrimp chitosan and chitosan extracted and purified from cell wall of the fungus were performed. Influences of different parameters on the biosorption of Cu²⁺ were studied. The Langmuir, Freundlich, Scatchard, Temkin, and Redlich-Peterson models were used to fit the equilibrium adsorption data. The adsorption rates were determined quantitatively and compared with pseudo-first order, pseudo-second order, intra-particle diffusion, and Elovich models.

Materials and methods

Shrimp chitosan

Low molecular weight shrimp chitosan with 60 cP viscosity (1% solution in 1% acetic acid), a product of BioLog Company Biotechnology and Logistics GmbH (Germany), was used in this research.

Fungal chitosan production and purification

Fungus M. indicus CCUG 22424 (Culture Collection, University of Göteborg, Sweden) was maintained on plates containing 40 g l^-1 glucose, 10 g l^-1 peptone, and 15 g l^-1 agar and kept at 32^ºC for 5 days to grow and then stored at 4^ºC until use. Liquid cultivation was carried out in a fermentor containing 90 g l^-1 date syrup (contained 7.2 % fructose, 7.0 % glucose, and 1.1% sucrose), 0.5 g l^-1 ammonium phosphate and 3 g l^-1 urea, and inoculated with 150 ml suspension containing 9 (±2) × 10⁶ spores ml^-1. The temperature was kept at 32^ºC and 2 vvm sterile air was introduced into the fermentor to provide aerobic conditions as well as mixing. The medium pH was adjusted at 5.0 to 5.5 using H₂SO₄ and NaOH. After complete consumption of the sugars (about 43 h), the fungal biomass was harvested on a screen and washed with distilled water until a clear filtrate appeared.

The fungal chitosan was separated and purified according to a recently developed method (17) with slight modifications. The harvested biomass was soaked in 0.5 M NaOH (30 ml NaOH per gram of dry biomass) and autoclaved for 20 min at 121^ºC to obtain the Alkali Insoluble Material (AIM). Afterwards, AIM was washed several times with distilled water to reach pH 7 and was finally dried at 50^ºC. Then, 2.5 g dried AIM was soaked and mixed for 15 min in 250 ml 72 mM H₂SO₄ to remove the phosphates. The residue was then washed with distilled water and soaked in 250 ml 72 mM H₂SO₄ and placed in an oil-bath at 120^ºC for 70 min. The liquid phase was poured into 300 ml 0.15 M NaOH in an ice bath and then centrifuged to extract the low molecular weight fungal chitosan. The chitosan was dried at 50^ºC.

Shrimp and fungal chitosan were ground and sieved to obtain particles with sizes less than 0.5 mm.

Degree of deacetylation

The Deacetylation degrees of shrimp and fungal chitosans were measured according to the method developed by Mohammadi et al. (18) which were both 85%.

Biosorption experiments

A stock solution containing 1000 mg l^-1 Cu²⁺ was prepared by dissolution of CuSO₄. 5H₂O in deionized water. The stock solution was diluted with deionized water to obtain solutions with desired concentrations. The solutions pH was adjusted using 0.1 M NaOH or 0.1 M H₂SO₄ before adding the chitosans. All experiments were carried out in 250 ml Erlenmeyer flasks containing 50 ml metal solution in a shaker-incubator at 135 rpm at certain temperatures. Control samples, without chitosan, with the same metal concentrations were also examined parallel to all tests. After reaching the equilibrium, the samples were centrifuged and concentration of the metal ions in the supernatant was measured.

Effect of initial pH of solution was examined by adding 0.03 g shrimp or fungal chitosan to a 50 ml Cu²⁺ solution (5 mg l^-1) at 32ºC. The experiments were carried out at pH 3.0, 3.5, 4.0, 4.5, 5.0, and 5.5. The mixtures were shaken to reach the equilibrium and the concentration of copper ions was measured.

Effects of chitosan loading were examined by addition of certain amounts of shrimp or fungal chitosan (0.01, 0.03, 0.05, 0.10, and 0.15 g) to 50 ml Cu²⁺ solutions (5 mg l^-1) at 32ºC and pH 4 and copper concentration was measured after equilibrium.

Effect of temperature on biosorption was examined by addition of 0.05 g shrimp or fungal chitosan to Cu²⁺ solution (50 ml) at pH 4 and different initial ions concentrations (5, 16, 28, 40, and 52 mg l^-1). The adsorption experiments were carried out at different constant temperatures of 13ºC, 32ºC, and 45ºC in a rotary shaker.

Effect of presence of chromium and lead ions on Cu²⁺ biosorption capacity of shrimp and fungal chitosan was also studied. Chitosan (0.03 g) was added to metal ionssolution (50 ml), containing 5 mg l^-1 of copper, chromium, and lead ions at pH 4 and 32ºC. Chromium and lead solutions were obtained by dissolution of K₂Cr₂O₇ and Pb (NO₃)₂ in deionized water, respectively.

In order to study the kinetics of biosorption, samples of shrimp or fungal chitosan (0.03 g) were added to a 50 ml Cu²⁺ solution (5 mg l^-1). The pH and temperature were set at 4 and 32ºC, respectively. The moment of chitosan addition to the solution was considered as time zero. Each data was achieved from individual flask at different times.

Adsorption isotherms were evaluated by addition of 0.05 g shrimp or fungal chitosan to Cu²⁺ solution (50 ml) at 32ºC and pH 4. Initial concentrations of the metal solutions were 5, 16, 28, 40, and 52 mg l^-1. In order to describe adsorption isotherm, Langmuir, Freundlich, Redlich-Peterson (19), Temkin (20), and Scatchard (21) models were used and the best model was identified. In all equations, q_e (mg g^-1) is adsorption capacity, which is the equilibrium amount of adsorbed solute per unit weight of chitosan, q_max (mg g^-1) is the amount of adsorbed ions to saturate unit mass of chitosan, and C_e (mg l^-1) is the equilibrium concentration of metal ions.

The Langmuir model is expressed as:

(1)

Where K (l mg^-1) is a constant related to the energy or net enthalpy of adsorption. The linear form of this equation is as follows:

(2)

Freundlich model is:

(3)

Where K_F ((mg/g) / (mg/l) ^1/n) and n show the capacity of adsorption and its intensity, respectively. The linear form of this model is: (4)

Temkin model is:

(5) )

Where A_T (l g^-1) and b_T (kJ mol^-1) are the model’s constants. R is the universal gas constant, and T (K) is the temperature.

Scatchard model is:

(6)

Where q_m (mg g^-1) and K_b (l mg^-1) are Scatchard model’s parameters.

Redlich-Peterson model is:

(7)

Where K_R (l g^-1), a_R (l mg^-1) ^β and β are the model’s parameters. Exponent of β is between 0 and 1. This equation can also be expressed as below:

(8)

Measurement of copper ions

Concentration of copper ions in all solutions was measured by an atomic absorption spectrometer (210 VGP, Buck Scientific Co. England). Adsorption capacity was calculated as:

(9)

Where V (l) is the volume of the solution which is 0.05 l in all experiments, m (g) is the mass of chitosan used, and C₀ and C_e(mg l^-1) are the concentration of copper ions before and after biosorption, respectively.

Statistical analysis

All experiments were carried out in duplicate and the average values were reported. Effect of chitosan type on adsorption capacity in all experiments was analyzed using PASW Statistics 18. T-test and analysis of variance (ANOVA) was performed at 5% level of significance.

Results

Kinetics of Cu²⁺ removal

In order to investigate the kinetics of biosorption, shrimp and fungal chitosans were added to a solution with 5 mg l^-1 copper ions. The concentration of ions and biosorption capacity of copper at different times are presented in Fig. 1(a) and (b), respectively. The results showed a very high initial rate of copper ions adsorption on both types of chitosan. Afterwards, the rate of biosorption decreased and finally, the system reached to equilibrium. The copper ions were removed under two steps, a rapid phase followed by a slower one (22) For shrimp chitosan, the adsorption capacity increased rapidly and 70% of initial copper ions adsorbed in the first 3 h and then decreased gradually to reach equilibrium in about 24 h. For the purified fungal chitosan, the equilibrium was reached after 3 h (Fig. 1).

In order to study the kinetics of adsorption, pseudo-first order (23), Ho’s pseudo-second order (23), intra-particle diffusion (24), and Elovich (23) models were evaluated. Ho’s model is the best model for description of kinetic data for shrimp and fungal chitosans, since correlation factors are higher than 0.99 (Table 1). Ho’s model is:

(10)

Table 1- Different models correlation factor (R²) and Ho’s model parameters for the biosorption of Cu²⁺ by shrimp and fungal chitosans

Fig. 1- Time distribution of copper ions concentration (a) and biosorption capacity (b) in solutions containing shrimp (●) and fungal (○) chitosans

where q_t (mgg^-1) is the amount of adsorbed metal ions per unit weight of adsorbent at time t (min), and K is the pseudo second-order reaction rate constant of adsorption (g mg^-1 min^-1). Linear regression analysis was used to fit the kinetic data to Ho’s model and q_e and K for both types of chitosan were calculated (Table 1). As this table demonstrates, Ho’s model is able to predict the kinetic data very well.

Effects of pH on the biosorption

Effects of initial solution pH, in the range of 3.0 to 5.5, on biosorption capacity for both types of chitosan were investigated (Fig. 2). The biosorption of copper by chitosan should be studied within the range of 3.0 - 5.5 since copper can precipitate as copper hydroxide at pH higher than 5.5. On the other hand, the chitosan can be dissolved in acidic solution at pH lower than 3 (25).

The biosorption capacity of chitosans was highly dependent on pH. With increasing the pH from 3.0 to 5.5, the biosorption capacity (q_e) increased from less than 2 to about 7.5 mg g^-1 for fungal chitosan and from less than 5 to more than 8 mg g,^-1 for shrimp chitosan. Analysis of variance showed that adsorption capacity is not dependent on chitosan type (Table 2)

Fig. 2- Effect of initial pH on biosorption of copper ion by shrimp (●) and fungal (○) chitosans

Table 2- Analysis of variance for dependence of adsorption capacity on chitosan type

Fig. 3- Effect of chitosan amount on equilibrium copper ion concentration (a) and biosorption capacity (b) for shrimp (●) and fungal (○) chitosans

Effects of adsorbent concentration

Effects of adsorbent concentration on Cu²⁺ removal were studied (Fig. 3(a) and (b)). As expected, the amount of adsorbed Cu²⁺ increased with increasing the amount of adsorbent. Increasing the amount of chitosan also resulted in lower adsorption capacity (q_e) for both shrimp and fungal chitosans.

Effects of temperature on biosorption

Effects of temperature on biosorption capacity of chitosans were investigated (Table 3). According to analysis of variance (Table 4), temperature did not have a significant effect on adsorption capacities of both shrimp and fungal chitosans. The adsorption capacity was slightly increased for shrimp chitosan with increasing the process temperature from 13ºC to 45ºC, while it was slightly decreased for the fungal chitosan. Besides, fungal chitosan has a slightly better adsorption capacity than shrimp chitosan at 13ºC.

Adsorption isotherm

Adsorption capacity as a function of equilibrium concentration of metal ions (C_e) is shown (Fig. 4). As can be seen, adsorption capacity increased with increasing the initial copper ions concentration. Furthermore, adsorption percentage decreased by increasing Cu²⁺ concentration (Fig. 5).

The parameters of the models for biosorption of Cu²⁺ by shrimp and fungal chitosans are calculated (Table 5). As correlation factors show (R²> 0.99), Langmuir, Freundlich, and Redlich-Peterson models accurately described the isotherm data for both types of chitosan.

Table 3- Effect of temperature on adsorption capacity of shrimp and fungal chitosans

Table 4- ANOVA for dependence of Adsorption capacity of shrimp and fungal chitosan on temperature

Fig. 4- Equilibrium biosorption of copper ions by shrimp (●) and fungal (○) chitosans

Fig. 5- Effect of copper ions concentration on adsorption percent by shrimp (●) and fungal (○) chitosans

Table 5 - Different models isotherm constants for biosorption of Cu²⁺ onto shrimp and fungal chitosans at 32ºC

Effects of presence of chromium and lead ions

The effects of chromium and lead ions on biosorption of Cu²⁺ by shrimp and fungal chitosans were also studied (Table 6). The results showed that adsorption capacity of copper ions did not considerably change in the presence of other ions for both types of chitosan. Thus, both types of chitosan can be successfully used for selective removal of copper ions from wastewaters and effluents containing the heavy metal ions.

Table 6- Biosorption of Cu²⁺ in a solution containing one, two and three metal ion(s)