EFFECT OF INCUBATION TIME ON COPPER UPTAKE AND BIOMASS OF ASPERGILLUS TERREUS تأثير وقت الحضانة على امتصاص النحاس والکتلة الحيوية لفطر الاسبريجلس تيروس

Effect of Incubation time on copper Uptake  and Biomass  of Aspergillus terreus

Sanaa Qaseem Badr¹ and Falah AbduIhasan Sahib²

(1)University of Basrah/ College of Marine Sciences / Dep. Natural Sciences / Iraq

(2)Minstry Agriculture / Iraq

ABSTRACT:

Fifty pure isolates were obtained from Aspergillus terreus which isolated from rivers at Basrah/Iraq to study the ability of fungus growth at different concentration of copper at different incubation periods. The capacity of the biological removal of copper was calculated by the fungus. The results showed that the rate of accumulation of the copper element in the fungus cells increases with the incubation period of the fungus with the element and the removal capacity of Aspergillus terreus  depend on the copper concentration and the time of incubation.

Key words: biological removal, accumulation, pollution, fungi, heavy metals.

INTRODUCTION:

Heavy metals pollution is becoming more serious problem for the environment and public health. Worldwide, heavy metals are transported via aerial, terrestrial, and aquatic systems [1]. Because of their persistence in the environment, heavy metals can accumulate in organismal tissues through the food chain. When heavy metals are not removed by an organism, especially toxic metals, they become toxic to that organism [2,3]. As a result, heavy metal pollution is a significant global problem. The input of heavy metals into aquatic ecosystems is affected by natural processes and anthropogenic activities, including geological weathering, mining effluents, industrial effluents, domestic effluents, rural nonpoint source pollution, and atmospheric deposition [1,4].  Association between different microbes has been used for wastewater treatment as bioremediations, as it was considered to have greater capability

over pure and single isolates. The axenic cultures were devised to be mineralized rapidly and completely by the metabolic activity of different microorganisms [5]. From microorganisms fungi have diverse mechanisms for bioremediation of heavy metals, including metal uptake by cell-wall components, accumulation inside the cell or extracellular chelation by phytochelatins and metal lothioneins [6].

The objective of the present study is to investigate the use of fungal biomass as the biosorbent for the removal of copper from an aqueous solution that contains copper sulfide nanoparticles.

MATERIAL AND METHODS:

• Sample collection and fungus isolation:

For the present study, water samples were collected from three river places at Basrah/Iraq.

The samples were cultured in a pure plate method and after examining the dishes and identified the fungus by usual methods. Fifty pure fungal isolates of Aspergillus terreus were used in the present study.

• Preparation of adsorbent:

Aspergillus terreus biomass was prepared in potato dextrose broth (PD broth) medium. For preparation of 100 ml of the PD broth, 30ml of potato broth and 2 gm of glucose was added in conical flask and completed to 100ml by distilled water. Flask was tightly closed with a cotton plug and aluminum foil and autoclaved at 121⁰C and 15 Pascal for 20 minutes.  Later, flask was opened under laminar flow and fungus was inoculated into each flask. The flasks were agitated on a rotatory shaker for 3-4 days at 150 rpm and at 30ºC temperature. After 3-4 days thick bed of fungal biomass developed was further used for biosorption experiment.

• Metal biosorption:

For investigation the biosorption capacity of Aspergillus terreus isolates towards the copper with various initial heavy metal concentrations and optimal cultural conditions. Copper solutions pH 5.0 of 11.07, 22.155 and 44.31mg/l were prepared using copper sulfate (CuSo₄.7H₂O). Then 1 ml of fungal biomass was suspended in 100 ml of copper solution in 250ml conical flask. The flasks were agitated on a rotatory shaker at 150rpm and at 30ºC with contact time of 24 hrs, 48 hrs and 72 hrs. The initial pH and biosorption contact time was chosen based on previous reported studies [7, 8].

• Equation calculating the proportion of compounds removed [9]

   R% = ((C0 – C1) / C) *100

Where: R = the percentage of removal.

C0 = the concentration of compounds in the primary solution.

C1 = the concentration of compounds in the final solution.

RESULTS AND DISCUSSION:

     Pollution by heavy metals affects the diversity of soil biota, their abundance and activity [10, 11]. When metals are discharged with effluent, they may result in severe contamination of downstream ecosystems [12]. In an aquatic environment, metals occur both in the dissolved or soluble fraction and in particulate matter. Elevated levels of metal ions are generally toxic and cause major damage to cells [13]. Interactions between different heavy metals and microorganisms might be antagonistic, additive or synergic. These interactions might be multifaceted and distinguished which depends on the type of heavy metal ion and microbial species consortium. The collective effect of different heavy metals may be toxic or growth enhancement in the same microbial consortium assorted from the additive effect of the single metal ions [14].  Biosorption technique employing microbial biomass has been illustrated. In this process both alive and heat killed dead biomass of several filamentous fungi (Mucor spp., Aspergillus spp., Penicillium spp., Rhizopus spp.) have been employed [15].

The results of the current study showed that the rate of accumulation of the copper element in the fungus cells was decreased with the incubation period of the fungus with the element at lower concentration as shown in Table 1.

Table 1: Average of bioaccumulation of the copper element in Aspergillus terreus

As the percentage of the biological removal of the copper component was calculated by the fungus, the results showed that the percentage of biological removal increases with the incubation period of the fungus with the polluted element as shown in Table 2

Table 2: Biomass removal rate of copper element by Aspergillus terreus

Capacity of fungi in biosorption of hazardous metals due to contain of fungi on chitin and lignin, exudation of organic acids by fungi or adsorption of metal ions to fungal cell wall [16,17]. This shows that the biosorption mechanism on fungal biomass surface is caused by interaction between heavy metals and different fungal species. The current results are similar to earlier studies which indicated the role of functional groups on fungal cell surface and the formation of complex bond among metal ions and various functional binding group [18,19].

Copper is rarely found in natural water, but is found in man polluted environments. Any copper present normally originates from industrial effluents, seepage, water from refuse dumps, pesticides or corrosive water that has come into contact with fitting and pipes containing copper [20,21]. Trace amounts of copper are essential for life, but it also catalyzes the synthesis of reactive oxygen species leading to severe damage to cytoplasmic constituents through oxidation of proteins, fragmentation of DNA and RNA, and lipid peroxidation [22].

 The results of biosorption vary from species to species because the process is dependent on factors including: fungal species, biosorbent size, metal solution concentration, solution pH,  shaking time and ionic composition. Fungi constitute a high proportion of the microbial biomass in soil. Being widespread in soil their large surface to volume ratio and high metabolic activity and can contribute significantly to heavy metal dynamics in soil [23].

REFERENCES:

[1] Förstner, U.; Wittmann, G.T. Metal Pollution in the Aquatic Environment, 2nd ed.; Springer: Berlin,Germany, 1981; pp.1–2, 30–58.

[2] Vukosav, P.; Mlakar, M.; Cukrov, N.; Kwokal, Ž.; Pižeta, I.; Pavlus, N.; Špoljaric, I.; Vurnek, M.;Brozincevic, A.; Omanovic, D. Heavy metal contents in water, sediment and fish in a karst aquaticecosystem of the Plitvice Lakes National Park (Croatia). Environ. Sci. Pollut. Res. 2014, 21, 3826–3839.

[3] Suresh, G.; Sutharsan, P.; Ramasamy, V.; Venkatachalapathy, R. Assessment of spatial distribution and potential ecological risk of the heavy metals in relation to granulometric contents of Veeranam lake sediments, India. Ecotox. Environ. Safe. 2012, 84, 117–124.

[4] DÍaz-de Alba, M.; Galindo-Riaño, M.D.; Casanueva-Marenco, M.J.; García-Vargas, M.; Kosore, C.M. Assessment of the metal pollution, potential toxicity and speciation of sediment from Algeciras Bay (South of Spain) using chemometric tools. J. Hazard. Mater. 2011, 190, 177–187.

[5] Lu, Z., Sun, X., Yang, Q., Li, H. and Li, C., Persistence and functionsof a decolorizing fungal consortium in a non-sterile biofilmreactor. Biochem. Eng. J., 2009, 46(1), 73–78.

[6] Cervantes, C. and Gutierrez-Corona, F., Copper resistance mechanismsin bacteria and fungi. FEMS Microbiol. Rev., 1994, 14(2),121–137.

[7] Kapoor A., Viraraghavan T. and Cullimore D.R., Removalof heavy metals using the fungus Aspergillus niger.Bioresour. Technol., 1999, 70, 95–104.

[8] Filipovic Kovacevic Z., Sipos L. and Briski F., Biosorption of chromium, copper, nickel and zinc ions onto fungal pellets of Aspergillus niger 405 from aqueous solutions. Food Technol. Biotechnol.,38 (3), 211–216 (2000)

[9] Qin, Y.; Shi, B. and Liu, J. 2006. j. chem. Technology . 13: 464-469p.

[10] Brookes, P.C.. The use of microbial parameters in monitoring soil pollution by heavy metals. Biol. Fertil. Soils, 1995,  19: 269-279.

[11] Inouhe, M., Sumiyoshi, M., Tohoyanna, H., Joho, M.. Resistance to cadmium ions and formation of a cadmium binding complex in various wild type yeasts. Plant Cell. Physiol., 1996,  37: 341-346.

[12] Nriagu, J.O. and PacynaA, J.M. Quantitative assessment of worldwide contamination of air, water and soils by trace metals. Nature, 1986, 333: 134-139

[13] Mhatre, G.N. ,  Pankhurst, C.E. Bioindicators to decontamination of soils with special reference to heavy metals. In: Biological indicators of soil health (eds. C.E. Pankhurst, B.M. Doube and V.V.S.R. Gupta), 1997, pp. 349-369. CAB International,Wallingford.

[14] Gias, P., Single and combined effects of nickel (Ni(II)) and cobalt (Co(II)) ions on activated sludge and on other aerobic microorganisms: a review. J. Hazard. Mater., 2008, 159(2), 187–203.

[15] Ahmad I., Ansari M.I. and Aqil F., Biosorption of Ni, Cr and Cd by metal tolerant Aspergillus niger and Penicillium spp. using single and multi-metal solution, Indian journal of experimental biology, 2006,  44, 73-76 .

[16] Akthar, M. N. and Mohan, P. M., Bioremediation of toxic metal ions from polluted lake waters and industrial effluents by fungal biosorbent. Curr. Sci., 1995, 69, 1028–1030.

[17] Bailey, S. E., Olin, T. J., Bricka, R. M. and Adrian, D. D., A review of potentially low-cost sorbents for heavy metals. Water Res., 1999, 33(11), 2469–2479.

[18] Galli, E., Di Mario, F., Rapana, P., Lorenzoni, P.  and Angelini, R.,Copper   biosorption by Auricularia polytricha. Lett. Appl.  Microbiol.2003,37(2),133–137.

[19] Luef, E., Prey, T. and Kubicek, C. P., Biosorption of zinc by fungal            mycelial wastes. Appl. Microbiol. Biotechnol., 1991, 34(5), 688–692.

[20] Udom, B.E., Mbagu, J.S.C., Adesodun, J.K., Aabim    N.N., 2004. Distributions of zinc, copper,cadmium and lead in a tropical   ultisol after long termdisposal of sewage sludge. Environ. Int., 30: 467- 470.  

[21] Andrews, S.; SutherlandD, R. A., 2004. Cu, Pb and Zn         contamination in Nuuanu watershed, Oahu, Hawaii.    Sci. Total Environ., 324: 173-182.

[22] Giaginis, C., Gatzidou, E., Theocharis, S.,2006. DNArepair systems as targets of cadmium toxicity. Toxicol. Appl.  Pharmacol., 213: 282-290.

[23]  Sosak-´Swiderska B., The soil fungi communities and risk assessment of         heavy metal contaminated soils management,Geophysical Research               , 12, 14357 (2010).

Effect of Incubation time on copper Uptake  and Biomass  of Aspergillus terreus

Sanaa Qaseem Badr¹ and Falah AbduIhasan Sahib²

(1)University of Basrah/ College of Marine Sciences / Dep. Natural Sciences / Iraq

(2)Minstry Agriculture / Iraq

ABSTRACT:

Fifty pure isolates were obtained from Aspergillus terreus which isolated from rivers at Basrah/Iraq to study the ability of fungus growth at different concentration of copper at different incubation periods. The capacity of the biological removal of copper was calculated by the fungus. The results showed that the rate of accumulation of the copper element in the fungus cells increases with the incubation period of the fungus with the element and the removal capacity of Aspergillus terreus  depend on the copper concentration and the time of incubation.

Key words: biological removal, accumulation, pollution, fungi, heavy metals.

INTRODUCTION:

Heavy metals pollution is becoming more serious problem for the environment and public health. Worldwide, heavy metals are transported via aerial, terrestrial, and aquatic systems [1]. Because of their persistence in the environment, heavy metals can accumulate in organismal tissues through the food chain. When heavy metals are not removed by an organism, especially toxic metals, they become toxic to that organism [2,3]. As a result, heavy metal pollution is a significant global problem. The input of heavy metals into aquatic ecosystems is affected by natural processes and anthropogenic activities, including geological weathering, mining effluents, industrial effluents, domestic effluents, rural nonpoint source pollution, and atmospheric deposition [1,4].  Association between different microbes has been used for wastewater treatment as bioremediations, as it was considered to have greater capability

over pure and single isolates. The axenic cultures were devised to be mineralized rapidly and completely by the metabolic activity of different microorganisms [5]. From microorganisms fungi have diverse mechanisms for bioremediation of heavy metals, including metal uptake by cell-wall components, accumulation inside the cell or extracellular chelation by phytochelatins and metal lothioneins [6].

The objective of the present study is to investigate the use of fungal biomass as the biosorbent for the removal of copper from an aqueous solution that contains copper sulfide nanoparticles.

MATERIAL AND METHODS:

• Sample collection and fungus isolation:

For the present study, water samples were collected from three river places at Basrah/Iraq.

The samples were cultured in a pure plate method and after examining the dishes and identified the fungus by usual methods. Fifty pure fungal isolates of Aspergillus terreus were used in the present study.

• Preparation of adsorbent:

Aspergillus terreus biomass was prepared in potato dextrose broth (PD broth) medium. For preparation of 100 ml of the PD broth, 30ml of potato broth and 2 gm of glucose was added in conical flask and completed to 100ml by distilled water. Flask was tightly closed with a cotton plug and aluminum foil and autoclaved at 121⁰C and 15 Pascal for 20 minutes.  Later, flask was opened under laminar flow and fungus was inoculated into each flask. The flasks were agitated on a rotatory shaker for 3-4 days at 150 rpm and at 30ºC temperature. After 3-4 days thick bed of fungal biomass developed was further used for biosorption experiment.

• Metal biosorption:

For investigation the biosorption capacity of Aspergillus terreus isolates towards the copper with various initial heavy metal concentrations and optimal cultural conditions. Copper solutions pH 5.0 of 11.07, 22.155 and 44.31mg/l were prepared using copper sulfate (CuSo₄.7H₂O). Then 1 ml of fungal biomass was suspended in 100 ml of copper solution in 250ml conical flask. The flasks were agitated on a rotatory shaker at 150rpm and at 30ºC with contact time of 24 hrs, 48 hrs and 72 hrs. The initial pH and biosorption contact time was chosen based on previous reported studies [7, 8].

• Equation calculating the proportion of compounds removed [9]

   R% = ((C0 – C1) / C) *100

Where: R = the percentage of removal.

C0 = the concentration of compounds in the primary solution.

C1 = the concentration of compounds in the final solution.

RESULTS AND DISCUSSION:

     Pollution by heavy metals affects the diversity of soil biota, their abundance and activity [10, 11]. When metals are discharged with effluent, they may result in severe contamination of downstream ecosystems [12]. In an aquatic environment, metals occur both in the dissolved or soluble fraction and in particulate matter. Elevated levels of metal ions are generally toxic and cause major damage to cells [13]. Interactions between different heavy metals and microorganisms might be antagonistic, additive or synergic. These interactions might be multifaceted and distinguished which depends on the type of heavy metal ion and microbial species consortium. The collective effect of different heavy metals may be toxic or growth enhancement in the same microbial consortium assorted from the additive effect of the single metal ions [14].  Biosorption technique employing microbial biomass has been illustrated. In this process both alive and heat killed dead biomass of several filamentous fungi (Mucor spp., Aspergillus spp., Penicillium spp., Rhizopus spp.) have been employed [15].

The results of the current study showed that the rate of accumulation of the copper element in the fungus cells was decreased with the incubation period of the fungus with the element at lower concentration as shown in Table 1.

Table 1: Average of bioaccumulation of the copper element in Aspergillus terreus

As the percentage of the biological removal of the copper component was calculated by the fungus, the results showed that the percentage of biological removal increases with the incubation period of the fungus with the polluted element as shown in Table 2

Table 2: Biomass removal rate of copper element by Aspergillus terreus

Capacity of fungi in biosorption of hazardous metals due to contain of fungi on chitin and lignin, exudation of organic acids by fungi or adsorption of metal ions to fungal cell wall [16,17]. This shows that the biosorption mechanism on fungal biomass surface is caused by interaction between heavy metals and different fungal species. The current results are similar to earlier studies which indicated the role of functional groups on fungal cell surface and the formation of complex bond among metal ions and various functional binding group [18,19].

Copper is rarely found in natural water, but is found in man polluted environments. Any copper present normally originates from industrial effluents, seepage, water from refuse dumps, pesticides or corrosive water that has come into contact with fitting and pipes containing copper [20,21]. Trace amounts of copper are essential for life, but it also catalyzes the synthesis of reactive oxygen species leading to severe damage to cytoplasmic constituents through oxidation of proteins, fragmentation of DNA and RNA, and lipid peroxidation [22].

 The results of biosorption vary from species to species because the process is dependent on factors including: fungal species, biosorbent size, metal solution concentration, solution pH,  shaking time and ionic composition. Fungi constitute a high proportion of the microbial biomass in soil. Being widespread in soil their large surface to volume ratio and high metabolic activity and can contribute significantly to heavy metal dynamics in soil [23].

REFERENCES:

[1] Förstner, U.; Wittmann, G.T. Metal Pollution in the Aquatic Environment, 2nd ed.; Springer: Berlin,Germany, 1981; pp.1–2, 30–58.

[2] Vukosav, P.; Mlakar, M.; Cukrov, N.; Kwokal, Ž.; Pižeta, I.; Pavlus, N.; Špoljaric, I.; Vurnek, M.;Brozincevic, A.; Omanovic, D. Heavy metal contents in water, sediment and fish in a karst aquaticecosystem of the Plitvice Lakes National Park (Croatia). Environ. Sci. Pollut. Res. 2014, 21, 3826–3839.

[3] Suresh, G.; Sutharsan, P.; Ramasamy, V.; Venkatachalapathy, R. Assessment of spatial distribution and potential ecological risk of the heavy metals in relation to granulometric contents of Veeranam lake sediments, India. Ecotox. Environ. Safe. 2012, 84, 117–124.

[4] DÍaz-de Alba, M.; Galindo-Riaño, M.D.; Casanueva-Marenco, M.J.; García-Vargas, M.; Kosore, C.M. Assessment of the metal pollution, potential toxicity and speciation of sediment from Algeciras Bay (South of Spain) using chemometric tools. J. Hazard. Mater. 2011, 190, 177–187.

[5] Lu, Z., Sun, X., Yang, Q., Li, H. and Li, C., Persistence and functionsof a decolorizing fungal consortium in a non-sterile biofilmreactor. Biochem. Eng. J., 2009, 46(1), 73–78.

[6] Cervantes, C. and Gutierrez-Corona, F., Copper resistance mechanismsin bacteria and fungi. FEMS Microbiol. Rev., 1994, 14(2),121–137.

[7] Kapoor A., Viraraghavan T. and Cullimore D.R., Removalof heavy metals using the fungus Aspergillus niger.Bioresour. Technol., 1999, 70, 95–104.

[8] Filipovic Kovacevic Z., Sipos L. and Briski F., Biosorption of chromium, copper, nickel and zinc ions onto fungal pellets of Aspergillus niger 405 from aqueous solutions. Food Technol. Biotechnol.,38 (3), 211–216 (2000)

[9] Qin, Y.; Shi, B. and Liu, J. 2006. j. chem. Technology . 13: 464-469p.

[10] Brookes, P.C.. The use of microbial parameters in monitoring soil pollution by heavy metals. Biol. Fertil. Soils, 1995,  19: 269-279.

[11] Inouhe, M., Sumiyoshi, M., Tohoyanna, H., Joho, M.. Resistance to cadmium ions and formation of a cadmium binding complex in various wild type yeasts. Plant Cell. Physiol., 1996,  37: 341-346.

[12] Nriagu, J.O. and PacynaA, J.M. Quantitative assessment of worldwide contamination of air, water and soils by trace metals. Nature, 1986, 333: 134-139

[13] Mhatre, G.N. ,  Pankhurst, C.E. Bioindicators to decontamination of soils with special reference to heavy metals. In: Biological indicators of soil health (eds. C.E. Pankhurst, B.M. Doube and V.V.S.R. Gupta), 1997, pp. 349-369. CAB International,Wallingford.

[14] Gias, P., Single and combined effects of nickel (Ni(II)) and cobalt (Co(II)) ions on activated sludge and on other aerobic microorganisms: a review. J. Hazard. Mater., 2008, 159(2), 187–203.

[15] Ahmad I., Ansari M.I. and Aqil F., Biosorption of Ni, Cr and Cd by metal tolerant Aspergillus niger and Penicillium spp. using single and multi-metal solution, Indian journal of experimental biology, 2006,  44, 73-76 .

[16] Akthar, M. N. and Mohan, P. M., Bioremediation of toxic metal ions from polluted lake waters and industrial effluents by fungal biosorbent. Curr. Sci., 1995, 69, 1028–1030.

[17] Bailey, S. E., Olin, T. J., Bricka, R. M. and Adrian, D. D., A review of potentially low-cost sorbents for heavy metals. Water Res., 1999, 33(11), 2469–2479.

[18] Galli, E., Di Mario, F., Rapana, P., Lorenzoni, P.  and Angelini, R.,Copper   biosorption by Auricularia polytricha. Lett. Appl.  Microbiol.2003,37(2),133–137.

[19] Luef, E., Prey, T. and Kubicek, C. P., Biosorption of zinc by fungal            mycelial wastes. Appl. Microbiol. Biotechnol., 1991, 34(5), 688–692.

[20] Udom, B.E., Mbagu, J.S.C., Adesodun, J.K., Aabim    N.N., 2004. Distributions of zinc, copper,cadmium and lead in a tropical   ultisol after long termdisposal of sewage sludge. Environ. Int., 30: 467- 470.  

[21] Andrews, S.; SutherlandD, R. A., 2004. Cu, Pb and Zn         contamination in Nuuanu watershed, Oahu, Hawaii.    Sci. Total Environ., 324: 173-182.

[22] Giaginis, C., Gatzidou, E., Theocharis, S.,2006. DNArepair systems as targets of cadmium toxicity. Toxicol. Appl.  Pharmacol., 213: 282-290.

[23]  Sosak-´Swiderska B., The soil fungi communities and risk assessment of         heavy metal contaminated soils management,Geophysical Research               , 12, 14357 (2010).