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Department of Botany, Damietta Faculty of Science, Mansoura University, New Damietta, Damietta Province, Egypt. P.O. Box 34517
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Thirty-two fungal species were isolated from a polluted watercourse near the Talkha fertilizer plant, Mansoura Province, Egypt. Aspergillus niger, A. flavus, Cunninghamella echinulata and Trichoderma koningii were isolated frequently. On the basis of its frequency, Cunninghamella echinulata was chosen for biosorption studies. Free and immobilized biomass of C. echinulata sequestered ions in this decreasing sequence is: Pb >Cu >Zn. The effects of biomass concentration, pH and time of contact were investigated. The level of ion uptake rose with increasing biomass. The maximum uptake for lead (45 mg/g), copper (20 mg/g) and zinc (18.8 mg/g) occurred at 200 mg/L biomass. The uptake rose with increasing pH up to 4 in the case of Pb and 5 in the case of Cu and Zn. Maximum uptake for all metals was achieved after 15 min. Ion uptake followed the Langmuir adsorption model, permitting the calculation of maximum uptake and affinity coefficients. Treatment of C. echinulata biomass with NaOH improved biosorbent capacity, as did immobilization with alginate. Immobilized biomass could be regenerated readily by treatment with dilute HCl. The biomass-alginate complex efficiently removed Pb, Zn and Cu from polluted water samples. Therefore,Cunninghamella echinulata could be employed either in free or immobilized form as a biosorbent of metal ions in waste water.
Key words: alkali treated, biosorption, copper, free and immobilized biomass, lead, zinc
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Venkateswerlu and Stotzky 1989, Luef et al 1991). Fungal biomass and seaweed biomass have been found to be excellent biosorbents for sequestering heavy metals (Lewis and Kiff 1988, Holan and Volesky 1994, Volesky and Holan 1995). Fungal biomass has been used to sequester copper, lead, zinc, nickel, cadmium, gold, silver and various actinide elements, such as thorium, uranium and plutonium (Tsezos and Volesky 1981, Gadd and White 1989, Luef et al 1991, Kapoor and Viraraghavan 1995, Meyer and Wallis 1997, Kapoor and Virraghavan 1998).
Fungi are known to have good metal uptake systems (Gadd 1986) with metabolism-independent bio-sorption being the most efficient. The specific mechanisms of uptake differ quantitatively and qualitatively according to the species, the origin of the biomass and its processing (Tobin et al 1984). The hyphal wall was found to be a primary site of metal ion accumulation (Tobin et al 1984). This is attributed to several chemical groups (the acetamido group of chitin; amino and phosphate groups in nucleic acids; amino, amido, sulfhydryl and carboxyl groups in proteins; and hydroxyls in polysaccharides) that might attract and sequester metal ions (Holan and Volesky 1995). Biomass of fungi, such as Absidia, Cunninghamella, Mucor and Rhizopus, exhibit excellent metalion uptake (Venkateswerlu and Stotzky 1989, Luef et al 1991, Fourest and Roux 1992, Mueler et al 1992). This could be due to the high chitin and chitosan content of the cell walls of these fungi (Tsezos and Volesky 1981).
The aim of this study was to isolate fungi from polluted water to find a new biosorbent agent. The ability of free and immobilized biomass of this biosorbent to sequester lead (Pb), copper (Cu) and zinc (Zn) was investigated.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) was used to concentrate fungal propagules from the sewage water. The fungal propagules were resuspended in sterilized bottles containing 50 or 100 mL of sterilized water, and 1 mL from each dilution was transferred to a clean sterilized Petri dish (three replicates). The culture medium contained aureomycin (0.35 g/mL), rose bengal (0.035 gm/mL), glucose (10 gm/L), peptone (5 gm/L) and agar (15 gm/L) (Cooke 1963). The medium was poured onto the samples within Petri dishes, and the plates were allowed to solidify. The plates were incubated at 28 C for 4–7 d. After incubation, species were isolated into pure culture and identified. The relative frequency of occurrence was calculated as the number of species isolated from each sample divided by the total number of samples. The isolated species were classified as very frequent (>20%), frequent (10–20%) or infrequent (<10%), as adapted from Tan and Leong (1989). Sporulating isolates were identified using various media for identification. Non-sporulating strains were grouped as mycelia sterilia according to similarities in colony morphology (Taylor et al 1999). Metal biosorption by free fungal biomass. – Cunninghamella echinulata was selected based on its frequent occurrence in sampling and on the fact that it has not been a subject in previous studies of this kind. The fungus was subcultured on PDA at 28 C for 7 d on a rotary shaker at 180 rpm. Fungal pellets were washed twice in sterile double-distilled water, drained and dried at 60 C to constant weight and ground with a mortar and pestle before determination of metal biosorption.
Effect of initial metal concentration. – To evaluate the effect of initial metal ion concentration (Ceq) on adsorption behavior of Cu, Zn and Pb by dried mycelial biomass, aliquots (50 mL) of 10, 50, 100, 200 and 300 mg/L concentrations of copper chloride, lead nitrate and zinc sulfate solutions were added to 100 mL Erlenmeyer flasks with a fixed biomass weight of 200 mg/L. The pH was adjusted to 4 with 0.1N HCl and 1N NaOH, and samples were mixed well by shaking. For sorption isotherm experiments, flasks were agitated on a rotary shaker (180 rpm) until no additional metal was removed (3–5 h). The samples were filtered through 0.45 µm millipore filters. Triplicate samples were analyzed by atomic absorption spectrophotometry. Samples also were taken from experimental controls, which contained no biomass.
Effect of biomass concentration. – To evaluate the effect of biomass concentration on the adsorption behavior of Pb, Cur and Zn, biomass concentrations of 50, 100 and 200 mg/L were added to 250 mL Erlenmeyer flasks separately. Aliquots (50 mL) of heavy metal solution (100, 200 and 300 mg/L) were added to each flask, and the flasks were left 15 min on a rotary shaker at 180 rpm (28 C) before being analyzed as above.
Effect of pH. – Adsorption of metal ions by dried mycelial biomass was studied at pH values of 2, 3, 4, 5 and 7. A fixed biomass of 200 mg/L was added to 50 mL of heavy metal solution containing Pb, Zn or Cu at an initial concentration of 200 mg/L for 15 min. To avoid shifts in pH due to biomass addition, the pH was adjusted with 0.1N HCl or 1N NaOH after the solution had been in contact with the adsorbent. In the case of Pb, pH was adjusted with 0.1N HNO3 or 0.1N NH4OH. Triplicate samples were analyzed as above.
Time of contact. – To determine the optimal incubation time, a fixed adsorbent concentration of 200 mg/L of fungal biomass was added to 50 mL of heavy metal solution containing an initial metal concentration of 200 mg/L of Pb, Zn and Cu. Three samples were taken at 5, 10, 15, 25, 35, 60 and 120 min and at 24 h and measured as above.
Biosorption mechanism. –
To determine the quantity of metal that can be attracted and retained in an "immobilized" form, it is customary to express metal uptake (q) by the biosorbent as the amount of metal adsorbed per unit of biomass. The calculation of the metal uptake (mg metal/g dry biosorbent) is based on the material balance of the sorption system. The amount of metal adsorbed by the biosorbent from solution can be estimated from this formula: q = v(Ci –Cf)/M (Holan and Volesky 1994), where q is the metal ion uptake, Ci is the initial concentration, Cf is the measured final concentration of the metal in solution, v is the liquid sample volume (mL) and M is the starting biosorbent weight (mg).
The sorption-isotherm relationship can be expressed mathematically by plotting q versus Cf. This first was done in the classical work of Langmuir (1918) who studied activated carbon adsorption. The linear form of the Langmuir isotherm equation is represented by this equation: q = Qmax bCf/1 + bCf, where Qmax is the maximum amount of metal per gram of biomass corresponding to saturation of the adsorption sites. The dissociation constant (b) is a coefficient related to the affinity between the metals and biomass.
Alkali treatment of the biosorbent. –
To generate ionic sites without significant modification of the cell wall structure before sorption, the mycelium was treated with 1 M NaOH (Fourest and Roux 1992). The dried and ground biosorbent was boiled in 0.1 M NaOH at 120 C for 6 h and filtered through 0.45 µm millipore filter paper. The treated mycelium then was washed several times to reach neutral pH and oven dried. After that, treated mycelium was added at 50, 100, 200 or 300 mg/L to 250 mL Erlenmeyer flasks. Aliquots (50 mL) of Pb, Zn and Cu (200 mg/L concentration) were added to each batch flask and left 15 min. The pH was adjusted to 4 in the case of Pb and 5 in the case Cu and Zn.
Metal biosorption by alkali-treated immobilized fungal biomass. – To each well of a percolating plate, 2 mg of alkali-treated biosorbent was added, followed by a drop of 4% sodium alginate of high viscosity. A drop of 0.25 M CaCl2 was added to each well separately to form beads. The beads were collected and air dried to yield pellets. Dried pellets, each containing 2 mg biosorbent biomass, were added to conical flasks to get final biomass concentrations of 20, 50 and 100 mg/L. A constant pH of 4 in the case of Pb and 5 in the case of Cu and Zn, and a time of contact of 15 min were used for all metal ions. After measuring the residual metal concentration in the solution, the beads were collected and regenerated using diluted acid (0.1 M HCl) followed by 3% bicarbonate. A control experiment was carried out using mycelium-free alginate-pellets.
Biosorption of Pb, Cu and Zn from polluted canal water. – A sample was collected from polluted effluents of the Talkha fertilizer factory, Mansoura Province. The sample contained 1, 2 and 1.5 mg/L of Pb, Cu and Zn, respectively. For removal of metal ions, immobilized fungal biomass was added to three beakers (five pellets each) containing 100 mL effluent without adjustment of pH. In a second experiment, the test was carried out at pH 4 and 5. After 15 min incubation, the residual metal concentration was determined by atomic absorption spectrophotometry.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). The majority of species were mitosporic (23 species of hyphomycetes and two species of agonomycetes). Zygomycota were represented by five species. The very frequent species were A. niger, followed by A. flavus, Cunninghamella echinulata and Trichoderma koningii.
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). Uptake decreased at 300 mg/L biomass (results not shown). Uptake rose with increase in pH up to 4 in the case of Pb and 5 in the case of Cu and Zn (FIG. 1b). The maximum uptake of all metals was achieved after 15 min, and this was followed by a decrease in the uptake (FIG. 1c).
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. Sorption isotherms represent the distribution of metal ions between aqueous and solid phases (biomass) when the concentration increases and as long as binding sites are not saturated. These isotherms permit the calculation of the adsorption capacities and dissociation constants of metal ions (TABLE II). Uptake (q200) decreased in this sequence: Pb (42.5 mg/g) >Cu (18.8 mg/g) >Zn (17.5 mg/g). The calculated Qmax is slightly higher than the experimental q200 (TABLE II).
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). The most removal of Pb, Cu and Zn were 95% (pH = 4), 95% and 89% (pH = 5) respectively.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, El-Morsy et al 2000). Of the species observed very frequently, only C. echinulata has not been tested as a biosorbent agent in previous studies (Meyer and Wallis 1997, Zahran 2002) and therefore was chosen as a subject for the studies reported here.
Biosorption by free biosorbent and affect of biomass concentration. –
Fourest and Roux (1992) reported that metalion uptake per gram of biosorbent increases as long as the biosorbent is not saturated. However, uptake values also depend on the nature and origin of the biosorbent itself (Luef et al 1991). In the present study, uptake of Pb, Cu and Zn by free biosorbent in solution varied depending on the initial metal concentration, biomass concentration, time of incubation and pH.
With C. echinulata, the optimal time for biosorption was 15 min after contact. This result is similar to that obtained by Kuyucak and Volesky (1989a, b) and Volesky and Philips (1995), who reported that most metal biosorption was achieved in 5–15 min, followed by residual and slower additional metal deposition (Tsezos and Volesky 1981), conceivably indicating a secondary metal binding mechanism.
Maximum uptake by C. echinulata biosorbent occurred at pH 4 for Pb and at pH 5 for Cu and Zn, with uptake falling with rising pH. The effects of pH on the biosorbent capabilities of fungal biomass appear to vary with assay conditions, the particular metal ion and fungal species. Similar to results reported here, a pH between 4 and 5 was reported as optimal for biosorption of Zn and Cu by Saccharomyces cerevisiae (Volesky and Phillips 1995); and Tobin et al (1984) and Tsezos and Volesky (1981) found that a pH near 4 was optimal for metal uptake by Rhizopus arrhizus. In contrast, Luef et al (1991) reported that biosorption of Zn by mycelium of A. niger, Penicillium chrysogenum and Clavicips paspali rose with increasing pH up to 9.0. Fourest and Roux (1992) reported that the optimal uptake of Zn and Pb by Rhizopus arrhizus was achieved at neutral pH and pH 5, respectively. Lewis and Kiff (1988) found that acidic pH reduced metal biosorption by the latter, a reduction that could be attributed to the precipitation of metal ions.
Biosorption mechanism. –
The sorption process involves biomass as a solid phase and a liquid phase containing metal ions, with ion distribution between solid and liquid phases determined by the affinity of biomass for metals. The quality of the biosorbent material is evaluated in terms of how much metal it can attract and retain in an immobilized form. It is customary to determine metal uptake (q) by the biosorbent as the amount of metals bound by the unit of biomass. Sorption isotherms follow the typical Langmiur adsorption pattern (Ruthven 1984). The results presented here for C. echinulata are consistent with the Langmuir-isotherm model (Fourest and Roux 1992).
Alkali treatment of biosorbent. –
Alkali treatment improved the capacity of the C. echinulata biosorbent to chelate metal ions, especially at the higher biomass of 200 mg/L. The higher affinity might be attributed to the chitin and chitosan content of the fungus cell wall, exposed after NaOH treatment. NaOH appears to remove amorphous polysaccharides from the cell wall, generating accessible space within the ß glucan-chitin skeleton and hence permitting metal ions to precipitate on this surface (Tsezos and Volesky 1981, Wainwright et al 1986, Luef et al 1991, Fourest and Roux 1992).
Higher (300 mg/L) and lower (50 mg/L) biosorbent concentrations exhibited reduced capacity for chelating ions. This reduction is attributable to a shortage in metal ions at higher biosorbent (300 mg/L) concentrations and to an excess at low biosorbent (50 mg/L) concentration, where all reactive sites on the cell wall are saturated with metal ions. These results argue against the hypothesis that electrostatic interaction between cells might be a significant factor in biomass-dependent metal adsorption (De Rome and Gadd 1987). Thus, increasing biosorbent concentration at a given metal concentration will not enhance the metal/biosorbent ratio and thereby the metal uptake per gram of biosorbent.
Immobilization biosorbent. –
Immobilization has been reported to enhance the capacity of fungal biomass for chelating metal ions (Luef et al 1991, Yousef 1997, Lewis and Kiff 1988). Here, alginate immobilization of alkali-treated biosorbent beads resulted in a nearly twofold increase in metalion uptake over free biosorbent. Alginate carboxyl groups are known to play an important role in metal binding, especially with cobalt (Kuyucak and Volesky 1989c).
Previous studies have revealed a high capacity of alginate-biomass beads to remove metal ions from polluted streams. In this study, up to 95%, 95% and 89% of Pb, Cu and Zn were removed, respectively. Kapoor and Viraraghavan (1998) used Aspergillus niger biomass immobilized in polysulfone polymer in the form of spherical beads to remove Cu, Pb and Zn ions from an industrial wastewater. A packed bed column containing Aspergillus niger beads removed 38% Cu, 58% Pb and 16% Zn from the culture in 12 h. Biomass immobilized on polyacrylamide similarly was reported to be superior to nonimmobilized biomass for metal biosorption (Yousef 1997).
Zygomycete species, such as Mucor spp., Rhizopus arrhizus, R. nigricans and Absidia orchidis generally are reported to be efficient biosorbent agents (Lewis and Kiff 1988, Fourest and Roux 1992, Mueler et al 1992, Holan and Volesky 1995). The work reported here demonstrates C. echinulata to be as effective as Mucor sp. and Rhizopus spp. but was less efficient than A. Orchidis, which adsorbed lead up to 351 mg/g (Holan and Volesky 1995).
In conclusion, biomass of C. echinulata efficiently is able to remove from solution Pb, Cu and Zn. This capacity was enhanced when the biomass was alkali treated and immobilized. The fungus in its immobilized form also was able to remove ions from a natural environment up to 95%. Therefore, the fungus is proposed as an effective biosorbent for removal of heavy metals in wastewater treatment.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 E-mail: el_morsy{at}mans.edu.eg 
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LITERATURE CITED |
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