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REMOVAL OF CHROMİUM, LEAD AND COPPER IONS FROM INDUSTRIAL WASTE WATERS BY Staphylococcus saprophyticus

Semra Ilhan1*, Macit Nurbaş Nourbakhsh2, Serpil Kiliçarslan2 and Huseyin Ozdag3

1Department of Biology, Osmangazi University, 26480 Eskisehir, Turkey 2Department of Chemical Engineering, Osmangazi University, 26480 Eskisehir, Turkey

3Department of Mining Engineering, Osmangazi University, 26480 Eskisehir, Turkey

ABSTRACT

In this study, selective biosorption of chromium, lead and copper ions by microorganisms from industrial wastewaters were investigated. Microorganisms were isolated from soil and in this research a bacterium which was identified as Staphylococcus saprophyticus was used. The effects of pH, temperature and initial concentration of metal ions on the biosorption capacity were investigated. The optimum pH values for chromium, lead and copper biosorption was found to be 2.0, 4.5 and 3.5 respectively. The maximum adsorption was observed for Cr6+, Pb2+and Cu2+, at the initial concentrations of 193.66 mg Cr6+/l; 100 mg Pb2+/l and 105 mg Cu2+/l and under these conditions the biosorption values were found to be 88.66 mg Cr6+/l; 100 mg Pb2+/l and 44.94 mg Cu2+/l, respectively.

The results indicated that S. saprophyticus was suitable for biosorption of lead and chromium from wastewaters.

Key words: Biosorption, adsorption, heavy metals, industrial wastewaters Staphylococcus saprophyticus. *Corresponding author. Tel.: +90 222 2290433/2419 fax: +90 222 2393578 E-mail address: silhan@ogu.edu.tr

INTRODUCTION

Metals are directly and/or indirectly involved in all aspects of microbial growth,

metabolism and differentiation. Many metals such as K, Na, Mg, Ca, Mn, Fe, Cu, Ni, Co, Zn,

Mo are essential for biological functions, whereas it is not known whether some others, such

as Al, Ag, Cd, Sn, Au, Sr, Hg, Tl and Pb have essential biological functions. All these

elements can interact with microbial cells and be accumulated as a result of physico-chemical

mechanisms and transport systems of varying specificity, independent on, or directly and

indirectly dependent on metabolism (Gadd, 1988). Some of these processes are of

biotechnological importance being relevant to metal removal and recovery from mineral

deposits and industrial effluents for industrial use or environmental bioremediation.

Turkish Electronic Journal of Biotechnology Vol 2, p:50-57, 2004 © Biotechnology Association

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Metals are introduced into aquatic systems as a result of the weathering of soils and

rocks, from volcanic eruptions, and from a variety of human activities involving the mining,

processing, or use of metals and/or substances that contain metal contaminants (Laws, 1993).

The last group which has the highest potential of toxicity and pollution includes waste waters

coming from metal plating industry, automobile, electrical and electronic materials, home

appliances, pipes, caps, guns, mechanics and dye industries. Unlike other pollutants, heavy

metals are getting important because of the fact that they can not be decomposed by in situ

biological means. Virtually all metals, including the essential metal micronutrients, are toxic

to aquatic organisms as well as humans if exposure levels are sufficiently high (Laws, 1993).

It is, therefore, necessary to remove heavy metals from waste waters before discharge due to

effects of disturbing environmental quality and being harmful to human health.

The removal of metals from industrial effluents can be achieved by ion exchange,

chemical oxidation, chemical precipitation etc (Aksu et al., 1992). For advanced purification,

different physico-chemical methods such as active carbon adsorption, ion exchange and

reverse osmosis are used. As an alternative to these methods, recently, the method of the

removal of heavy metal contaminants by means of bacteria has been focused on. Biological

removal of heavy metal contaminants from aquatic effluents offers great potential when

metals are present in trace amounts (Fourest and Roux, 1992; Vinita and Radhanath, 1992).

Many microbial species such as bacteria, fungi, yeast and algae are known to be

capable of adsorbing heavy metals on their surface and/or accumulating within their structure

(Campbell and Martin, 1990; Luef, 1991; Mitani and Misic, 1991; Vinita and Radhanath,

1992;). It is possible that microorganisms can be used in the removal of toxic metal ions from

the water and even in the recovery of them by using these adsorption properties of the

microorganisms. Physical adsorption or ion exchange at the living or non-living cell surface is

very rapid and occur in a short time after microorganisms come into contact with heavy metal

ions. Accumulation occurs in living cells and is slow, related to metabolic activity (Gadd,

1990; Gadd, 1992, Nourbakhsh at al., 2002). Although there are a number of studies on

removal of heavy metals, the knowledge of the applications of biosorbents in the environment

and industry has not been clear yet.

In this study, some important parameters that should be considered in the removal of

heavy metals from the industrial waste waters were investigated and an approach concerning

with application to the industrial waste water containing heavy metals have been presented.

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MATERIALS AND METHODS

Isolation and identification of microorganisms:

The bacterium strain used in this study was isolated from the soil samples undergone

enrichment process (Nourbakhsh at al., 2002). The enrichment was carried out in the shaken

culture of the medium with the following contents: 4.0 g/l D(+) glycose monohydrat, 0.8 g/l

KH2PO4, 4.0 g/l (NH4)2SO4, 0.1 g/l NaH2PO4, 0.2 g/l MgSO4.7H2O, 4.0 g/l yeast extract, 0.1

g/l CaSO4.H2O, 4.0 g/l pepton, 4.0 g/l nutrient broth, 0.1 g/l FeCl3.6H2O, 0.1 g/l

Na2MoO4.2H2O. Pure culture was obtained by inoculation to nutrient plates from the

enrichment culture. Isolates were investigated with respect to their removing capacity of lead,

copper and chromium from the solutions containing these heavy metal ions. The isolate

OGUB 003 was selected. Microscopic and biochemical tests were applied to this isolate

according to Bergey’s Manual of Systematic Bacteriology and API 20E (Schleifer, 1986). The

genus to which the isolate belongs was determined.

Growth of Microorganisms and Biosorption

OGUB 003 was incubated at 27°C and at 150 rpm for 40-48 hours in tripticase soy

broth (Oxoid). At the end of incubation, biomass was separated from medium by centrifuging

at 5000 rpm and it was kept in the oven at 50ºC to remove the free water as much as possible.

Then, it was suspended in deionized water in order to use it in the biosorption. 100 ml

solutions containing 100 mg/l Cr6+, 150 mg/l Pb2+, and 100 mg/l Cu2+ were prepared from

stock solution containing 1 g/l Cr6+ (K2Cr2O7), 1 g/l Pb2+ (Pb(NO3)2), 1 g/l Cu2+ (CuSO4).

Then, 4.0 g microorganism (gmo) were added to the medium (20 gmo/l) and adsorptions of

metals were investigated for different pH values adjusted by using HCl and NaOH at 27°C.

The solution containing the biomass was agitated in a shaker of 150 rpm during the

adsorption. Samples taken at predetermined intervals were centrifuged and supernatants were

analyzed. The analyses of Cr6+, Pb2+, and Cu2+ ions were carried out by atomic absorption

spectrophotometer (Perkin-Elmer) at 0.01 ppm sensitivity level after dilution of the samples.

By taking the determined optimum conditions into consideration, the capacity of

microorganism to remove the mentioned metals from an industrial waste water was searched

with the same method.

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RESULT AND DISCUSSION

The isolate OGUB 003 was identified as Staphylococcus saprophyticus. First, for

biosorption of Cr6+, Pb2+ and Cu2+ ions by wet biomass of OGUB 003 optimum conditions

(pH, temperature, ion concentration) were determined. The highest biosorption of Cr6+ was

determined at pH 2, temperature 27ºC, initial ion concentration 193.66 mg/l while for Pb2+

this was realized at pH 4.5, temperature 27 ºC, initial ion concentration 100-150 mg/l. For the

Cu2+ optimum adsorption conditions were determined to be pH 3.5, temperature 27ºC, 105

mg/l initial ion concentration. Obtained results are shown in Table 1, 2, 3 and 4.

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Under the optimum conditions determined (Table 4) the highest uptake, %100 yield,

was determined for lead ions. Biosorption of the metals in their individual solutions by S.

saprophyticus was determined to be Pb2+>> Cr6+ >Cu2+. The walls of Gram positive bacteria

are efficient metal chelators and in Bacillus subtilis, the carboxyl group of the glutamic acid

of peptidoglycan was the major site of metal deposition. Teichoic and teichuronic acids were

important binding sites in Bacillus licheniformis (Gadd, 1990). S. saprophyticus is a Gram

positive bacterium and it has similar cell wall properties as of other Gram-positive bacteria.

The study was focused on biosorption of lead ions due to the high recovery rate of lead

ions by S. saprophyticus. Because lead ions were fully biosorbed from the solution having a

100 mg/l Pb2+ initial concentration, experiments were carried out on the solution of 250 mg/l

Pb2+ initial concentration, in order to apply the process to waste waters containing higher lead

ion concentration. The equilibrium time was determined to be 2 hours because after 2 hours

the adsorption rate was insignificant. However it was observed that the uptake reached to 70%

during the first 15 min. This high uptake can be attributed to physical adsorption on the cell

wall consisting of peptidoglucan (Gadd, 1990). Figure 1 shows the metal ratio biosorbed.

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The waste water samples taken from a plating unit in Eskişehir were used as industrial waste

water. The results of removal of Cr6+, Pb2+ and Cu2+ ions from an industrial waste water by S.

saprophyticus were given in Table 5.

While in the individual solutions the biosorption yield of the metals by S.

saprophyticus was 100%, 24,2% and 14,5% for Pb2+, Cr6+ and Cu2+, respectively, in a mixed

solution these were 100 %, 25% and 24 % for Pb2+, Cr6+ and Cu2+. Because the adsoption was

selective (Nakajima and Akaguchi, 1986) and high in the industrial waste waters having low

lead ion concentrations (0.17 and 0.47 mg/l Pb2+), it can be said that S. saprophyticus can be

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used in tertiary treatment of the waste water containing lead ions at low concentrations (Vinita

and Radhanath, 1992).

As a result, it can be concluded that S. saprophyticus can be used in the removal or recovery

of heavy metal ions, especially lead ions from industrial waste waters. It can also be said that

the usage of S. saprophyticus can be successful to remove chromium element from the waste

waters containing higher levels of chromium ions. Further studies are needed to increase the

biosorption capacities of biomass and to develop appropriate technologies applicable in the

treatment of industrial waste waters.

Acknowledgments

Authors acknowledge the Research Foundation of Osmangazi University for support

the project as numbered 1997/15.

REFERENCES

Aksu, Zümriye; Sağ Yeşim and Kutsal Tülin. The biosorption of Copper (II) by C.

vulgaris and Z. ramigera. Environmental Technology, 1992, vol.13, p. 579-586.

Campbell, R. and Martin, M. H. Continuous flow fermentation to purify wastewater

by the removal cadmium. Water, Air and Soil Pollution, 1990, vol. 50, p. 397-408.

Fourest, E. and Roux, J. C. Heavy metal biosorption by fungal mycelial by-products:

mechanism and influence of pH. Applied Microbiology and Biotechnology, 1992, vol. 37, p.

399-403,.

Gadd, Geoffrey Micheal. Biotechnology-a comprehensive treatise Weinheim; VCH

Verlagsgesellschaft, 1988, p. 401-433.

Gadd, Geoffrey Micheal. Heavy metal accumulation by bacteria and other

microorganisms. Experientia, 1990, vol. 46, Birhauser Verlag, CH-4010, Basel Switzerland.

Gadd, Geoffrey Micheal. Metals and microorganisms: a problem of definition. FEMS

Microbiology Letters, 1992, vol. 100, p. 197-204.

Laws, E. A., Aquatic Pollution. Second ed. John Willey and Sons Inc. 1993. p: 351-

415.

57

Luef, E.; Prey, T. and Kubicek, C. P. Biosorption of zinc by fungal mycelial wastes.

Applied Mivrobiology and Biotechnology, 1991, vol. 34, p. 688-692.

Mitani, T. and Misic, D. M. Copper Accumulation by Penicillium sp. Isolated from

soil. Soil Sci. Plant Nutr. 1991, vol. 37, no. 2, p. 347-349.

Nakajima, A. and Akaguchi, T. Selective accumulation of heavy metals by

microorganisms, Applied Microbiology and Biotechnology, 1986, vol. 24, p. 59-64.

Nourbakhsh, Nurbaş Macit; Kiliçarslan, Serpil; Ilhan, Semra and Ozdağ, Hüseyin.

Biosorption of Cr6+, Pb2+ and Cu2+ ions in industrial waste water on Bacillus sp. Chemical

Engineering Journal, 2002, vol. 85, p. 351-355.

Schleifer, Karl Heinz. Bergey’s Manual of Systematic Bacteriology, London, Williams

and Wilkins, 1986. 1599 p. ISBN 0-683-04108-8 (v.1).

Vinita, V. P. and Radhanath, P. D. Biorecovery of zinc from industrial effluent using

native microflora. Intern. J. Enviromental Studies, 1992 , vol. 44, p. 251-257.