Adsorption of copper metal by living Aspergillus niger L. biomass

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adsorption of copper metal on living biomass of Aspergillus niger was investigated. 100 mg .... 50 ml of 10ppm solution of copper was taken in each of a set of 6 flasks. Similarly ..... textile dyes from aqueous solutions by dead fungal biomass.

INTERNATIONAL JOURNAL OF ENVIRONMENTAL SCIENCES Volume 5, No 6, 2015 © Copyright by the authors - Licensee IPA- Under Creative Commons license 3.0 Research article

ISSN 0976 – 4402

Adsorption of copper metal by living Aspergillus niger L. biomass Preeti Gupta, Pooja, Pradip Kumar and Nidhi Singh Department of Biotechnology, C.C.S. University, Meerut and BASE Association, Shastri nagar, Meerut [email protected] doi: 10.6088/ijes.2014050100106 ABSTRACT Water pollution is the contamination of water bodies e.g. lakes, rivers, oceans, aquifers and groundwater. It occurs when pollutants are discharged directly or indirectly into water bodies without adequate treatment to remove detrimental compounds. Among the pollutants which are discharged into water bodies, heavy metals are of most concern because other pollutants may be degraded by some microorganisms but metals cannot be degraded. In this study, adsorption of copper metal on living biomass of Aspergillus niger was investigated. 100 mg and 200 mg doses of living Aspergillus niger biomass and 10 minute contact time showed the most appropriate amount and duration for use in biosorption of copper effluent. Maximum adsorption capacity of 100 mg living biomass with respect to different concentrations of copper solution was observed as 95.66%, 91.33%, 63.91%, and 21.26% at 10ppm, 20ppm, 40ppm and 80 ppm respectively. In case of 200 mg biomass, as much as 98.43% metal removal was observed from the higher metal concentration (80ppm), The biosorption was gradually increased, but exceptionally, a negligible reduction was observed in biosorption from 65.95% at 10ppm to 51.72% at 20ppm. The adsorption isotherm studies indicate that Freundlich model is more suitable for describing the biosorption of 100 mg and 200 mg living Aspergillus niger biomass. The kinetic studies indicated that the biosorption process follows a pseudo-first order model for both the doses of biomass of Aspergillus niger for Cu metal. Key words: Dyes, wastewater, textile effluents, biosorption, Langmuir and Fruendlich 1. Introduction Environmental contamination is a major problem being faced by the society today. Heavy metal removal from wastewater is important for the protection of the environment and human health. One of the more important toxic metals, Copper, is essential to human life and health, but like all heavy metals, it is potentially toxic, especially at high concentrations (Tapiero et al., 2003), Copper finds its way to the water stream from industries like electoplating, mining, electrical/ electronics, iron and steel production, also in the non-ferrous metal industry, the printing & photographic industries, metal working and finishing processes (Birlik et al., 2006), Copper may cause serious health problems, in particular possible liver damage with prolonged exposure. Copper in water sources can also damage a variety of fish and invertebrates. Large acute doses can produce harmful, even fatal effects. These reasons suggest that, copper must be removed to very low levels from wastewater (Turkmen et al., 2009), Biosorption of heavy metals from aqueous solutions is a relatively new technology for the treatment of wastewater (Schiewer and Volesky, 2000), Absorbent materials (biosorbents),

Received on January 2015 Published on May 2015

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derived from a suitable biomass, can be used for the effective removal and recovery of heavy metallic ions from wastewater streams. The biosorption does not replace methodologies used for removal of metals (such as precipitation, reduction, ion exchange, adsorption and coagulation); however, it can act as a system polishing in processes that are not completely efficient. Treatment of effluents with heavy metals following biotechnological approaches is simple, comparatively inexpensive and environment friendly (Han et al., 2006; Pal et al., 2006; Vijayaraghavan et al., 2006), Microbiological processes are having great significance in determining metal mobility and have potential application in bioremediation of metal pollution (Umrania, 2006), Microbial biomass can be used to decontaminate metal bearing wastewaters as well as to concentrate metals. The nature of biological surfaces is such that different functional groups form complexes with metal ions (Huang et al., 1991), resulting in chemical complex as an uptake mechanism. In previous studies, one of the fungus was capable of removing metals from a number of substrates is Aspergillus niger. This filamentous fungus is widely used in the fermentation industry because of its ease of culturing and lack of pathogenicity to humans and animals (Berka et al., 1992), A. niger has been used to remove metals from the environment by either adsorption of the metals to fungal cell wall components, or complexation of the metals with organic acids produced by the fungus (Akthar and Mohan, 1995; bosshard et al., 1996), Thus, the main objectives of this study were to quantify: (a) the ability of living Aspergillus niger biomass to biosorb copper metal solutions; (b) the effect of different concentrations of Cu (as CuSO4) metal solution on the biosorptive capacity of living A. niger biomass (c) with the effect of different doses of living A. niger biomass on biosorption of cu metal solutions. (d) Comparative assessment of adsorption isotherms (langmuir and fruendlich) (e) and of adsorption kinetics (pseudo first order reaction and pseudo second order reaction), 2. Material and methods Approximately, 1kg of soils were collected (apparently free from pollution) from agriculture fields near Meerut-Hapur bypass, Meerut City (U.P.), The upper layer of the soil was removed with the help of a trowel to remove extraneous litter/organic matter. Soil samples were then taken out with the help of a trowel aseptically in fresh sterile polythene bags. The samples were analyzed for soil mycobiota using dilution plate method (Waksman, 1927), 2.1 Preparation of fungal biomass Aspergillus niger van Tieghem isolated from the soil were selected for studies on biosorption of copper (II), Axenic culture of Aspergillus niger was prepared on Potato Dextrose Agar plate. For preparing the biomass of the selected fungal strain, MGYP broth medium was prepared. The composition of the broth medium is as under: Malt extract (3 gm), Glucose (10 gms), Yeast extract (3gms), Pepton (5 gms), Distilled water (1000 ml), pH (6.8), 2.2. Preparation of copper solution of different concentration Copper metal (Copper sulphate) was used to assess the ability of the fungal biomass to adsorb metal. Stock solution of heavy metal was prepared in a manner so as to obtain different concentrations i.e., 10ppm, 20ppm, 40ppm, 80ppm solution of metal. 3. Biosorption of copper effluents using living fungal biomass Preeti Gupta et al., International Journal of Environmental Sciences Volume 5 No.6 2015

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50 ml of 10ppm solution of copper was taken in each of a set of 6 flasks. Similarly, a set of 6 flasks were used for each of the three concentrations (i.e. 20ppm, 40ppm, 80ppm) of copper. To these flasks, wet fungal biomass was added as under: The method was followed by Bhole et al. (2004) was adopted with slight modification to determine the sorption profile of the Cu (as CuSO4) using of test fungi i.e. living Aspergillus niger biomass. 1. 10ppm CuSO4 solution (control i.e. without biomass)- (3 flasks) 2. 10ppm CuSO4 solution + 100 mg biomass (3 flasks) 3. 10ppm CuSO4 solution + 200 mg biomass (3 flasks) 4. 20ppm CuSO4 solution (control i.e. without biomass)- (3 flasks) 5. 20ppm CuSO4 solution +100 mg biomass (3 flasks) 6. 20ppm CuSO4 solution + 200 mg biomass (3 flasks) 7. 40ppm CuSO4 solution (control i.e. without biomass)- (3 flasks) 8. 40ppm CuSO4 solution +100 mg biomass- (3 flasks) 9. 40ppm CuSO4 solution + 200 mg biomass (3 flasks) 10. 80ppm CuSO4 solution (control i.e. without biomass)- (3 flasks) 11. 80 ppm CuSO4 solution +100 mg biomass- (3 flasks) 12. 80ppm CuSO4 solution + 200 mg biomass (3 flasks) All these flasks were then placed on a rotatory shaker for 10 minutes. After a contact period of 10 minutes, the fungal biomass was separated by filtering the mixture through Whatman No. 40 filter paper to prevent the probable interference of turbidity and the filtrate was further processed for assessing the concentration of copper remaining in the solution. The content of 3 flask of each set were pooled together to get a composite solution for visible spectrophotometery. The concentration of remaining copper in the supernatant was assessed with the help of visible spectrophotometer at wavelength of 645-650 nm. Biosorbent + 100 ml Metal solution Shaking for 10 min. on rotatory shaker Filtered through Whatman No- 40 filter paper Save the supernatant Take O.D. at 645-650nm The specific uptake (Q-value) of copper was calculated as follows: Q = V(Ci-Cf)/ m Where, Q= copper uptake (mg copper / g biosorbent), V= the liquid sample volume (ml), Ci = the initial concentration of the copper in solution, Cf = the final concentration of copper in the solution, m = the amount of added biosorbent on the dry basis (mg) Similarly, biosorption efficiency (R%) of particular biomass can be calculated as under: R = [ (Ci-Cf)/Ci ] × 100% The biosorptive efficiency of particular biomass of test fungus was interpreted as under: Preeti Gupta et al., International Journal of Environmental Sciences Volume 5 No.6 2015

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1. 2. 3. 4. 5.

0-10 10-20 20-40 40-60 60-80

Very poor Poor Moderate Good Very good

The empirical models viz., Freundlich (1906) and Langmuir (1918) for single solute system and modified Langmuir for multiple situations were employed to biosorption equilibria of the test fungus. (1) Freundlich equation: qe=KFCe1⁄n Langmuir equation: qe=

(2)

Where, qe= metallic ions absorbed per unit weight of adsorbent at equilibrium (mg/g); qmax = maximum amount of metallic ions adsorbed per unit of weight of adsorbents (mg/g); KL= constant related to the affinity of binding sites for metal ions (L/mg); Ce = equilibrium (residual) concentration (mg/L); KF = Freundlich characteristic constant of the system, incorporating parameters affecting the adsorption process, such as adsorption capacity; n = Freundlich characteristic concentration on the adsorption on the system, incorporating parameters such as effecting of concentration on the adsorption capacity and represents the adsorption intensity (dimensionless), 4. Adsorption kinetics Langergren’s kinetics equation (1898) has been most widely used for the adsorption of an adsorbate from an aqueous solution. Lagergren summarized the pseudo-first order rate equation for the liquid-solid adsorption system as follows: Log (qe-qt) = loge t (3) Where; qe = mass of metal adsorbed at equilibrium (mg/g); qt = adsorbed amount at equilibrium; K1 = rate constant in the pseudo-first-order (min); t = time (min), The pseudo-second-order model (Ho and McKay, 1999) was followed for second order kinetics analysis. The linear form can be written as follows: = + t (4) Where; qe = the mass of dye adsorbed at equilibrium (mg/g); qt = the mass of metal at time t (min); K2 = rate constant in the pseudo-second-order (g/ mg min); t = time (min), 5. Results and discussion The present study was conducted to as such the capability of biomass of living Aspergillus niger to adsorb copper metal from the solutions of different concentrations of copper (as Copper Sulphate) metal. Different amounts of living fungal biomass as biosorbents i.e. 100 Preeti Gupta et al., International Journal of Environmental Sciences Volume 5 No.6 2015

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mg and 200 mg were allowed to biosorb the metal from solutions of test metal solutions. The observations were taken after ten minutes of contact time period. The results obtained are presented in the Table 1. It reveals that living Aspergillus niger biomass is quite effective for the biosorption of copper metal solutions. As much as 95.66% could be adsorbed in 100 mg biomass after reaction of ten minutes contact period at higher concentration of metal in solution i.e., 80ppm. While at lower concentrations, from 10ppm to 40ppm, the biosorption of copper by test fungal biomass increased effectively from 21.26% at 10ppm, 63.91% at 20ppm and 91.33% at 40ppm metal (Table 1, Fig.1), Increase in test fungal biomass from 100 mg to 200 mg led to increase in biosorption percentage up to 98.43%. The maximum Q-value (Table 1, Fig.1) was observed (in case of 100 mg biomass) at 80ppm metal concentration i.e., 38.26, followed by 18.26 at 40ppm, 6.39 at 20ppm and 1.06 at 10ppm metal concentrations. In case of 200 mg biomass, the pattern of increase in biosorption was observed as in case of 100 mg of test biomass. As much as 98.43% metal removal was observed from the higher metal concentration (80ppm) by 200 mg biomass. The biosorption was gradually increased, but exceptionally, a negligible reduction was observed in biosorption from 65.95% at 10ppm to 51.72% at 20ppm. Further increase in metal concentration had a positive influence on the biosorption and led to increase in biosorption efficiency from 51.72% at 20ppm to 90.31% at 40ppm metal concentration. The maximum Q-value (Table 1, Fig.2) was observed (in case of 200 mg biomass) at 80ppm metal concentration i.e., 19.68, followed by 9.03 at 40ppm, 2.58 at 20ppm and 1.64 at 10ppm metal concentrations. At a glance, the Q-value (Table1, Fig.2) reveals that increase in biomass caused a great decrease in the specific uptake of metal per milligram of biomass.Kumar (2011) resulted that the initial concentrations of lead and chromium in the solution have a marked influence on the rate of sorption of these metals by fungal biomass and it was also noted that with increase in the initial concentration of metal ions, their adsorption also increased at a great extend.

Figure 1: (a) Cu biosorption (b) Cu bisorption % profile of living Aspergillus niger biomass (100 mg) Thus, it is clear that the selection of amount of biomass for the biosorption of all the copper concentrations of test metal at a particular incubation period can be adjusted without much effect that the use of 100 mg biomass was most appropriate concentration for use in the present case for specific metal uptake. Chauhan et al. (2002) reported that the presence of higher concentration of biomass in the solution result in reduced distance between biosorbent particles. Thus, many binding sites remain unoccupied. Asma et al. (2006), Farah et al. (2007), Jaikumar and Ramamurthi (2009) and Farah and El-Gendy (2012) concluded that the Preeti Gupta et al., International Journal of Environmental Sciences Volume 5 No.6 2015

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biosorption capacity (mg/g) decreased with increase in biosorbent dosage, due to the agglomeration of the adsorbent particles. However, further detailed study on saturation kinetics of metal biosorption is required through further light on this issue barring one exception. It was clearly indicate that as the biosorbent concentration increase saturation point was reached early.

Figure 2: (a) Cu biosorption (b) Cu biosorption % profile of living Aspergillus niger biomass (200 mg) Reaz and Charaya (2008) resulted that with every dose of biosorbent a saturation point is reached after a certain contact time period beyond which the biosorbent particles seem to release biosorbent molecules back into the effluents (desorption) and this may be followed by resorption. Horsefall et al. (2003) suggested that sorption of dye molecules on the biomass is a reversible process. Both adsorption and desorption process are likely to occur. Initially, the high concentration of dye molecules in the solution and the dye molecules occupy the binding sites of the biosorbent. However, after reaching a saturation point, the dye molecules are released back into the solution followed by possible resorption. The probability of existence of such a situation is more only when a multilayered structure exists. The adsorption isotherm models to the observations recorded in the present study are presented in the Fig. 3 and Fig. 4. A perusal of these figures reveals that Freundlich model is more suitable for describing the biosorption of 100 mg and 200 mg living Aspergillus niger biomass in the studies concern ranges. Much better correlation coefficients 0.803 and 0.487 were obtained using Freundlich isotherm model as compare to 0.508 and 0.323 using Langmuir isotherm model in case of 100 mg and 200 mg living A. niger biomass, respectively. Zhang et al., 2011 concluded that the biosorption of Cu(II) by biosorption membrane of Penicillium biomass followed the Langmuir isotherm while Gazem and Nazareth (2012) concluded that the biosorption of Cu(II) using the fungus Aspergillus versicolor followed the Freundlich’s isotherm. The Langmuir isotherm model (Langmuir, 1918) predicts the formation of adsorbed solute monolayer with no side interactions between the adsorbed ions. It also assumes that the Preeti Gupta et al., International Journal of Environmental Sciences Volume 5 No.6 2015

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interactions taken place by adsorption of one ion per binding site and also that the sorbent surface is homogenous containing only one type of binding site. On the other hand, the Freundlich’s model (Freundlich, 1906) assumes the existence of multilayered structure (Fourest and Volesky, 1996), As per this model, the energetic distribution of this site is heterogeneous. As per the earlier discussion, the results of the present study better fit into the Freundlich’s model. Therefore, it provides support to the conclusion regarding sorption and desorption in the preceding paragraph. A comparison of between two kinetic models suggested (Figures 5 and 6) that the coefficient of correlation (r2) for the pseudo-first order kinetic model is much higher in comparison to pseudo-second order kinetic model in the case of both the doses of biomass of Aspergillus niger for Cu metal. Velkova et al. (2012) concluded that the biosorption of Cu(II) from aqueous solution using waste mycelium of industrial xylanase-producing strain Aspergillus awamori followed the pseudo-first-order kinetic reaction while Huang et al. (2012) concluded that the biosorption of Cu(II) fruiting body of jelly fungus (Auricularia polytricha) from aqueous solutions followed the pseudo-second-order kinetic reaction. Table 1: Biosorption of copper (as CuSO4) from aqueous solutions of different concentrations by living biomass of Aspergillus niger L. Amount of biomass (gm)

100 mg

200 mg

Initial concentration of Cu in the solution (ppm)

Final concentration of Cu in the solution (ppm)

Amount of Cu adsorbed (ppm)

Percentage of Cu adsorbed

Q-value

10 20 40 80 10 20 40 80

7.874 7.218 3.468 3.468 3.408 9.655 3.874 1.248

2.126 12.782 36.532 76.532 6.595 10.345 36.126 78.751

21.260% 63.91% 91.33% 95.66% 65.950% 51.725% 90.315% 98.438%

1.063 6.39 18.266 38.266 1.6487 2.5862 9.0315 19.6877

Figure 4: (a) Freundlich’s isotherm: sorption profile of Aspergillus niger (100 mg biomass) for Cu metal Preeti Gupta et al., International Journal of Environmental Sciences Volume 5 No.6 2015

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Figure 4: (b) Freundlich’s isotherm: sorption profile of Aspergillus niger (200 mg biomass) for Cu metal Table 2: Amount of bio mass absorption Amount of biomass

N

1/n

Kf

R2

100 mg

3.835827

0.2607

8.85

0.8034

200 mg

1.946283

0.5138

8.323

0.4874

Figure 3: (a) Langmuir’s isotherm: sorption profile of Aspergillus niger (100 mg biomass) for Cu metal

Figure 3: (b) Langmuir’s isotherm: sorption profile of Aspergillus niger (200 mg biomass) for Cu metal Preeti Gupta et al., International Journal of Environmental Sciences Volume 5 No.6 2015

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Table 3: Amount of bio mass absorption Amount of biomass 100 mg

a

b

1/ab

R2

0.147

0.9407

7.2317

0.508

200 mg

0.669

0.6065

2.4648

0.323

Figure 5: (a) Psuedo-first-order kinetics of copper solution (100 mg living Aspergillus niger biomass)

Figure 5: (b) Pseudo-second order kinetics of copper solution (100 mg living Aspergillus niger biomass)

Figure 6: (a) Psuedo-first-order kinetics of copper solution (200 mg living Aspergillus niger biomass) Preeti Gupta et al., International Journal of Environmental Sciences Volume 5 No.6 2015

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Figure 6: (b) Pseudo-second order kinetics of copper solution (200 mg living Aspergillus niger biomass) 5.1 Conclusion The above results revealed that Aspergillus niger biomass is a very good biosorbent of Cu metal and can be used for adsorption based effluent treatment system e.g. in heavy metal treatment systems. In the present study, the maximum dose (200 mg) observed the most appropriate concentration for use in biosorption of Cu metal (as CuSO4 solution), Thus the biosorption capacity increases with the increasing dose of biomass. Biosorption of Cu metal by Aspergillus niger biomass is a cost-effective and green technology. The Aspergillus niger biomass proved a major component for the development of biotrap devices and it may be used as a major component for managing the heavy metal pollution in our country. Conventional technologies to clean up heavy metal from industrial effluents are time taking and less effective. An alternative to these technologies, biosorption is inexpensive, fast and promosing solution towards bio-detoxification of heavy metal pollution from the wastewaters. 6. References 1. Asma D., Kaharaman S., Ching S., Yesilada O., (2006), Adsorptive removal of textile dyes from aqueous solutions by dead fungal biomass. Journal of Basic Microbiology, 46, pp 3-9. 2. Bhole B.D., Genguly B., Madhuram A., Deshpande D., Joshi J., (2004), Biosorption of methyl violet, basic fuchsin and their mixture using dead fungal biomass. Current Science, 86, pp 1641-1644. 3. Birlik E., Ersoz A., Denizli A., Say R., (2006), Preconcentration of copper using double-imprinted polymer via solid phase extraction. Analytica Chimica Acta, 565, pp 151. 4. Chauhan P., Mohan M., Sarangi R.K., Kumari B., Nayak S.R., Matandkar S.G.P., (2002), Surface chlorophyll a estimation in the Arabian sea using IRS−P4 Ocean colour monitor (OCM) satellite data. International Journal of Remote Sensing, 23, pp 663-676. 5. Farah J.Y., and El-Gendy N.S., (2012), Performance, kinetics and equilibrium in biosorption of anionic dye Acid Red 14 by the waste biomass of Saccharomyces

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cerevisiae as a low-cost biosorbent. Turkish Journal of Engineering and Environmental Sciences, 1, pp 1-6. 6. Farah J.Y., El-Gendy N.S., Farahat L.A., (2007), Biosorption of astrazone blue basic dye from an aqueous solution using dried biomass of Baker’s yeast. Journal of Hazardous Materials, 148(1−2), pp 402-408. 7. Fourest E., and Volesky B., (1996), Contribution of sulfonate groups and alginate to heavy metal biosorption by the dry mass of Sargassum fluitans. Environmental Science and Technology, 30, pp 277-282. 8. Freundlich H.M.F., (1906), Over the adsorption in solution. Journal of Physical Chemistry, 57, pp 385-470. 9. Gazem M.A., and Nazareth S., (2012), Isotherm and kinetic models and cell surface analysis for determination of the mechanism of metal sorption by Aspergillus versicolor. World Journal of Microbiology and Biotechnology, 28(7), pp 2521-2530. 10. Han R., Zhang J., Zou W., Xiao H., Shi J., Liu H., (2006), Biosorption of copper (II) and lead (II) aqueous solution by chaff in a fixed bed column. Journal of Hazardous Materials, B133, pp 262-268. 11. Ho Y.S., and McKay G., (1998), Kinetic models for the sorption of dye from aqueous solution by wood. Process Safety and Environmental Protection, 76, 183191. 12. Horsefall M., Abia A.A., Spitt A.J., (2003), Removal of Cu(II) and Zn(II) ions from waste water by cassava (Mannihot esculenta Cranz.) waste biomass. African Journal of Biotechnology, 10, pp 360-369. 13. Huang H., Cao L., Wan Y., Zhang R., Wang W., (2012), Biosorption behavior and mechanism of heavy metals by the fruiting body of jelly fungus (Auricularia polytricha) from aqueous solutions. Applied Microbiology and Biotechnology, 96(3), pp 829-8240. 14. Jaikumar V., and Ramamurthi V., (2009), Effect of biosorption parameters kinetics isotherm and thermodynamics for acid green dye biosorption from aqueous solution by brewery waste. Internantional Journal of Chemistry, 1, pp 2-12. 15. Kumar P., (2011), Studies on Certain Biotechnological Aspects of Microbe-Metal Interactions. Ph.D. Thesis, C.C.S. University, Meerut, India. 16. Langmuir I., (1918), The adsorption gasses on plane surface of glass, mica and platinum. Journal of the American Chemical Society, 40, pp 1361-1368. 17. Pal A., Ghosh S., Paul A.K., (2006), Biosorption of cobalt by fungi from serpentine soil of Andaman. Bioresource Technology, 97, pp 1253-1258.

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18. Reaz A.W., and Charaya M.U., (2008), Adsorption of Violet Dye by Spent Tea Leaves Powder from Industrial Effluents. National Academy Science Letters, 31(1−2), pp 23-26. 19. Schiewer S., and Volesky B., (2000), Biosorption processes for heavy metal removal. In: Lovley DR, editor. Environmental microbe– metal interactions. Washington, DC: ASM Press, pp 329-357. 20. Tapiero H., Townsend D.M., Tew K.D., (2003), Trace elements in human physiology and pathology. Copper. Biomedicine & Pharmacotherapy, 57 (9), pp 386. 21. Turkmen D., Yılmaz E., Ozturk N., Akgo S., Denizli A., (2009), Poly(hydroxyethyl methacrylate) nanobeads containing imidazole groups for removal of copper(II) ions. Materials Science and Engineering, 29(6), pp 2072. 22. Umrania V.V., (2006), Bioremediation of toxic heavy metals using acidothermophilic autotrophes. Bioresource Technology, 97, pp 1237-1242. 23. Velkova Z., Stoytcheva M., Gochev V., (2012), Biosorption of Cu(II) onto chemically modified waste mycelium of Aspergillus awamori: Equilibrium, kinetics and modeling studies. Journal of Bioscience and Biotechnology, 1(2), pp 163-169. 24. Vijayaraghavan K., Palanivelu K., Velan M., (2006), Biosorption of copper (II) and cobalt (II) from aqueous solutions by crab shell particles. Bioresource Technology, 97, pp 1411-1419. 25. Zhang X., Su H., Tan T., Xiao G., (2011), Study of thermodynamics and dynamics of removing Cu(II) by biosorption membrane of Penicillium biomass. Journal of Hazardous Material, 193, pp 1-9.

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