1. Introduction

Heavy metals are on the forefront of academic and regulatory concern, since millions of gallon of water containing toxic heavy metals are generated annually from several metal processing industries and discharged into the environment. Metals thus discharged into water bodies are not readily biodegraded but thus undergoes chemical or microbial transformations, creating large impact on the environment and public health ^[1].

Several conventional wastewater technologies have been developed and are successfully in use at a large scale, in order to reduce hazardous concentration of compounds in wastewater to lower and safe levels ^[2]. The need for effective and economical methods for removing heavy metals from waste water has resulted in the search for material that may be useful in reducing the levels of accumulation of heavy metals in the environment ^[3]. Microbial populations in metal polluted environments include microorganisms which have adapted to toxic concentrations of heavy metals and become metal resistant ^[4]. Biomaterials like algae, fungi, bacteria and activated sludge have been tested as biosorbents for heavy metal removal ^{[5, 6, 7]}. Iron a common contaminant enters the water bodies through effluents of industries such as electroplating, mining, steel processing etc. The presence of Fe (II) in water results in undesirable color, odour and taste which makes water unfit for industry and domestic consumption. The aim of the present work was to study the removal of iron from solutions and synthesis of iron nanoparticles using a strain of Aspergillus species isolated from soil samples collected from metal plating industry.

2. Materials and Methods

Aspergillus species isolated from the soil samples collected from Hyderabad metal plating industry, I.D.A, Balanagar, Hyderabad, A.P. were preserved at 4°C in a refrigerator.

The isolated pure culture of Aspergillus species was grown in fungal media containing KH₂PO₄–7.0 g/L,KH₂PO₄–2.0 g/L, MgSO₄ · 7H₂O– 0.1 g/L, (NH₄)₂SO₄– 1.0g/L, yeast extract–6.0 g/L, glucose–10 g/L) with different concentrations of of FeSO_{4 .}5H₂O (2, 4, 6,8,10 and12mM) and a set of broth culture devoid of iron sulphate served as a control. Growth curves were obtained using dry mass weight of fungal growth in different concentrations against time variation.

Aspergillus species grown in different concentration of metal were harvested during early stationary phase. To the 50ml filtrate obtained after filtration add 2ml nitric acid. Amount of iron present in the filtrate was analyzed by Atomic Absorption Spectroscopy (GBC Scientific Equipments Ltd).

Aspergillus sp. was grown in 1mM Ferrous sulphate and after 48hrs. The medium was centrifuged and fungal pellet was collected. The fungal pellet was used for TEM studies to identify the localisation of metal and to know the Aspergillus sp. Capacity to synthesize iron nanoparticles.

3. Results and Discussion

The organism isolated from the soil sample was identified by amplication of the 18s rRNA. The isolated Aspergillus sp. from the soil sample collected from industrial area is an indication that the microorganisms are capable of growing in the presence of high concentrations of heavy metals like iron.

Figure 1. Growth kinetics of Aspergillus species in absence and presence of various concentrations of iron sulphate

Download as

PowerPoint Slide

Larger image(png format)

Figures index

Veiw figure

View current figure in a new window

View next figure

The growth kinetics (Figure 1) of Aspergillus species grown in iron sulphate showed the fungi growing in lower concentration of iron showed increased log phase as compared to high concentration of iron. Lag phase was not observed when the fungi was grown in different concentrations of iron. The presence of iron was found to decrease the growth of Aspergillus sp as compared to the growth in the media without iron. As the external iron concentration is increased the growth of the organism is not affected. This may be due to decreased the bioavailability of the toxic metals/metalloids through extracellular complexation, precipitation, and binding to cell wall constituents ^[8].

Figure 2. The amount of iron present in the medium

Download as

PowerPoint Slide

Larger image(png format)

Figures index

Veiw figure

View current figure in a new window

View previous figure

View next figure

As shown in (Figure 2) Atomic Absorption Spectrophotometer analysis shows that the amount of metal in the medium is more at 3.0 mM range and afterwards it is decreased. As the iron concentration in the broth was increased the sequestration of iron on to the cell wall increased. Iron is an essential metal and requires a proper uptake, intracellular transport and storage to avoid the deleterious consequences of the appearance of free iron in either the cytosol or the cell organelles. In filamentous fungi, intracellular siderophores like ferricrocin and hydroxyferricrocin have been reported to keep excess iron in a thermodynamically inert state ^{[9, 10, 11]}.

But TEM observations of Aspergillus sp. cells exposed to iron solution showed electron dense metal nanoparticles are adsorbed on the cell walls. (Figure 3). The size of the nanoparticles are in range of 50-200nm. As reported ^[8] ferrous ions may bound to the cellwall to regulate metal stress. Guerinot stated that microbes have developed various strategies for acquiring iron and at the same time protecting themselves from potential toxic effects of iron ^[12]. Beveridge and Murray ^[13] related the bacterial metal interactions to the anionic character of specific functional groups situtated on membrane components (e.g. teichoic acids, peptidoglycan, phospholipids). A. Ferrooxidans growing in iron rich environment accumulated uranium on the cell wall ^[14]. Brown precipitates of Fe(III) adhering to the hyphae surface in cultures of A. alternata (SA), C. cladosporoides (TO), and Ph. Glomerata (SA) are reported ^[15].

Figure 3. Transmission electron micrograph of Aspergillus sp. grown in iron sulphate

Download as

PowerPoint Slide

Larger image(png format)

Figures index

Veiw figure

View current figure in a new window

View previous figure

4. Conclusion

This study has shown that the iron can be removed from waste water using Aspergillus sp. Hence Aspergillus species can be considered for use in Biological filter systems for removal of iron present in industrial effluents and Waste water. Considering the wide array of molecular genetic tools available in genetic engineering modified, Aspergillus strains be created for use in future in the versatile metal removal technologies and Aspergillus based nanoparticle producing technologies is foreseeable.

Acknowledgement

This research was financially supported by Gokaraju Rangaraju Institute of Engineering and Technology, Hyderabad, India. We would like to thank Dr. Laksham of Sri Venkateswara Veterinary University for his technical assistance during TEM observations.

References

[1]	Volesky. B. and Holan, Z, “Biosorption of heavy metals”, Biotechnol. Prog, 11, 235-250, 1995.
	In article		CrossRef PubMed
[2]	Verma, N. and Rehal, R, “Removal of Chromium by Albizia libbeck pods from industrial wastewater”, J. ind. Pollut. Control, 12, 1, 55-59, 1996.
	In article
[3]	Egwaikhide, P.A. Asia, 1.O. and Emua, S.A, “Binding of Zinc, Nickel and Cadmium ions by carbonized Rice husk”, African. J. Sci, 3,2, 673-681, 2002.
	In article
[4]	Kasan, H.C. and Baeckert, A.A.W, “Activated sludge treatment of coal gasification effluent in a petrochemical plant. Metal accumulation by heterotrophic bacteria”, Water Sci. Tech. 21,4/5, 297-303, 1989.
	In article
[5]	Lodi, A. Solisoio, C. Converti, A. and DelBorghi, “Cadmium, Zinc, Copper, Silver and Chromium (III).Removal from waste waters by Sphaerotilus natans”, Bioprocess Eng. 19,3, 197- 203,1989.
	In article
[6]	Taniguchietal, J. Hemmi, H. Tanahashi, K. Amano,N.Nakayama, T. Nishim, T. 2000. Zinc biosorption by a zinc resistant bacterium Brevibacterium sp. Strain, H2M-1. Appl Microbial Biotechnol. 54, 581-588.
	In article		CrossRef
[7]	Valdman,E and Leite S.G.F,“Biosorption of Cd, Zn and Cu by Saragssum Sp. Waste biomass”, Bioprocess Eng, 22, 171-173,2000.
	In article		CrossRef
[8]	González-Guerrero, M. Benabdellah, K. Ferrol, N. and Azcón-Aguilar, C, Mechanisms underlying heavy metal tolerance in arbuscular mycorrhizas. In: Azcón-Aguilar C, Barea JM, Gianinazzi S, Gianinazzi-Pearson V (eds) Mycorrhizas—Functional Processes and Ecological Impacts. Springer, Berlin, 107-122, 2009.
	In article		CrossRef
[9]	Eisendle, M. Schrettl, M. Kragl, C. Müller, D. Illmer, P. and Haas, H, “The intracellular Siderophore ferricrocin is involved in iron storage, oxidative-stress resistance, germination, and sexual development in Aspergillus nidulans”, Eukaryot Cell, 5,10, 1596-1603,2006.
	In article		CrossRef PubMed
[10]	Schrettl, M. Kim, H.S. Eisendle, M. Krag,l C. Nierman, W.C, Heinekamp, T. Werner, E.R. Jacobsen, I. Illmer, P. Yi, H. Brakhage, A.A. Haas, H, “SreA-mediated iron regulation in Aspergillus fumigates”, Mol Microbiol, 70, 1, 27-43,2008.
	In article		CrossRef PubMed
[11]	Johnson, L, “Iron and siderophores in fungal-host interactions”, Mycol Res, 112,170-183, 2008.
	In article		CrossRef PubMed
[12]	Guerinot, M.L, “Microbial iron transport”, Annu. Rev. Microbial, 48,1, 743-772, 1994.
	In article		CrossRef PubMed
[13]	Beveridge,T.J. and Murray,R, “Sites of metal deposition in the cell wall of Bacillus subtilis”, Journal of Bacteriology, 141, 2, 876-887, 1980.
	In article		PubMed
[14]	Johnson, D.B, “Biodiversity and ecology of acidophilic microorganisms”, FEMS Microbiology Ecology, 27, 4, 307-317, 1998.
	In article		CrossRef
[15]	De la Torre, M.A. and Gomez-Alarcon,G, “Manganese and Iron Oxidation by Fungi Isolated from building Stone”, Microb Ecol, 27,2,177-188, 1994.
	In article		CrossRef