INTERNATIONAL JOURNAL OF ENVIRONMENTAL SCIENCES Volume 2, No 2, 2011

© Copyright 2010 All rights reserved Integrated Publishing Association

Research article ISSN 0976 – 4402

Removal of Heavy Metals from Industrial Effluent Using Bacteria

Manisha Nanda1 , Dinesh Sharma2 , Arun Kumar3
1, 2- Department of Zoology and Biotechnology, H.N.B. Garhwal Central University
Srinagar,Garhwal (UK), Badshahi Thol Campus Tehri Garhwal (UK) India
3- Department of Biotechnology, Dolphin (PG) Institute of Biomedical and Natural Sciences,
Dehra Dun (UK) India
[email protected]
doi:10.6088/ijes.00202020038

ABSTRACT

Industrial development results in the generation of industrial effluents, and if untreated results
in water, sediment and soil pollution. (Fakayode and Onianwa, 2002; Fakayode, 2005).
Industrial wastes and emission contain toxic and hazardous substances, most of which are
detrimental to human health (Jimena et al.,2008; Ogunfowokan et al.,2005; Rajaram et
al.,2008). The key pollutants include heavy metals, chemical wastes and oil spills etc. Heavy
metal resistant bacteria have significant role in bioremediation of heavy metals in wastewater.
The objective of this work is to study the role of bacteria in removing the heavy metals
present in the industrial effluent.Five effluent samples out of nine were selected for this study
due to high content of heavy metals. The heavy metals Hg and Cu were removed by Bacillus
sp. The average Hg reduction was 45% and Cu reduction was recorded as 62%. The heavy
metals Cd, As and Co were removed by Pseudomonas sp. The average Cd reduction was
56%, average As reduction was 34% and average Co reduction was recorded as 53%. The
heavy metals Cd and Cu were removed by Staphylococcus sp. The average Cd reduction was
44% and average Cu reduction was recorded as 34% .

Keywords: Heavy Metal, industrial effluent, cadmium, arsenic, mercury.

1. Introduction

Rapid industrial development have led to the recognition and increasing understanding of
interrelationship between pollution, public health and environment. Industrial development
results in the generation of industrial effluents, and if untreated results in water, sediment and
soil pollution. (Fakayode and Onianwa, 2002; Fakayode, 2005). Industrial wastes and
emission contain toxic and hazardous substances, most of which are detrimental to human
health (Jimena et al.,2008; Ogunfowokan et al.,2005; Rajaram et al.,2008).

Heavy metals from industrial processes are of special concern because they produce water or
chronic poisoning in aquatic animals (Ellis, 1989).While some heavy metals are purely toxic
with no cellular role (Shi et al.,2002),other metals are essential for life at low concentration
but become toxic at high concentrations (Badar et al. 2000), high concentration of all heavy
metals inhibhits activity of sensitive enzymes (Koropatnick and Leibbrandt 1995).Heavy
metals can damage the cell membranes, alter enzymes specificity, disrupt cellular functions
and damage the structure of the DNA. Toxicity of these heavy metals occurs through the
displacement of essential metals from their native binding sites or through ligand interactions.
Also, toxicity can occur as a result of alterations in the conformational structure of the nucleic
acids and proteins and interference with oxidative phosphorylation and osmotic balance.

Received on September 2011 Published on November 2011 789

 Removal of Heavy Metals from Industrial Effluent Using Bacteria

Heavy metals are not biodegradable and tend to be accumulated in organisms and cause
numerous diseases and disorders (Ozer and Pirincci, 2006).

Conventional physicochemical methods such as electrochemical treatment, ion exchange,

precipitation, reverse osmosis, evaporation, and sorption (Kadirvelu et al.,2001(a); Kadirvelu
et al.,2002) for heavy metal removalfrom waste streams are not cost effective (Kadirvelu et
al.,2001(b)) and hence biological approach has been considered as an alternative remediation
for heavy metal contamination.To survive under metal-stressed conditions, bacteria have
evolved several types of mechanisms to tolerate the uptake of heavy metal ions. These
mechanisms include the efflux of metal ions outside the cell, accumulation and complexation
of the metal ions inside the cell, and reduction of the heavy metal ions to a less toxic state
(Nies, 1999). The complex structure of microorganisms implies that there are many ways for
the metal to be taken up by the microbial cell. The biosorption mechanisms are various and
are not fully understood. They may be classified according to various criteria. According to
the dependence on the cell's metabolism, biosorption mechanisms can be divided into:

1. Metabolism dependent; and

2. Non -metabolism dependent.

According to the location where the metal removed from solution is found, biosorption can
be classified as:

1. Extra cellular accumulation/ precipitation;

2. Cell surface sorption/ precipitation; and
3. Intracellular accumulation.

Transport of the metal across the cell membrane yields intracellular accumulation, which is
dependent on the cell's metabolism. This means that this kind of biosorption may take place
only with viable cells. It is often associated with an active defence system of the
microorganism, which reacts in the presence of toxic metal. During non-metabolism
dependent biosorption, metal uptake is by physico-chemical interaction between the metal
and the functional groups present on the microbial cell surface. This is based on physical
adsorption, ion exchange and chemical sorption, which is not dependent on the cells'
metabolism.

Cell walls of microbial biomass, mainly composed of polysaccharides, proteins and lipids
have abundant metal binding groups such as carboxyl, sulphate, phosphate and amino groups.
This type of biosorption, i.e., non-metabolism dependent is relatively rapid and can be
reversible (Kuyucak and Volesky, 1988). In the case of precipitation, the metal uptake may
take place both in the solution and on the cell surface (Ercole et al., 1994). Further, it may be
dependent on the cell's' metabolism if, in the presence of toxic metals, the microorganism
produces compounds that favour the precipitation process. Precipitation may not be
dependent on the cells' metabolism, if it occurs after a chemical interaction between the metal
and cell surface.

2. Materials and Method

2.1 Sample Collection

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 Removal of Heavy Metals from Industrial Effluent Using Bacteria

Industrial waste water samples were collected from a nearby pharmaceutical industrial area in
Dehradun in sterile plastic bottles and were brought to the lab aseptically. The samples were
stored at 4° C for further use.

2.2 Isolation of metal resistant bacteria

In the present study heavy metal resistant bacterial species were isolated from the soil and
waste water by serial dilution and pour plating method using Nutrient agar supplemented
with different heavy metal salts.Strains were maintained in agar slants containing nutrient
agar. They were characterized morphologically and on the basis of biochemical reactions.
They were transferred weekly to new medium in order to keep metabolic activity and
checked for purity by microscopic examination.

2.3 Exposure of bacteria to heavy metals

Bacteria were cultured in nutrient broth supplemented with different heavy metal metal salts
and the concentration of the metal salts was maintained at 1mM of the medium. Cells were
inoculated in nutrient broth (100 mL/flask) and kept under agitation in a rotary shaker, at 80
rpm, for 48 hours at 35 ± 2ºC.Cells to be used in biosorption experiments were separated by
centrifugation.

2.4 Measurement of Physico-Chemical parameters of the effluent samples

The physico-chemical parameters were measured in all samples pH were measured by digital
pH meter. DO, BOD and COD was measured by titration method.

2.5 Determination of Heavy Metals concentration in industrial Effluent

The estimation of heavy metals was done by Inductive coupled plasma mass spectrometry
(ICP-MS).

2.6 Treatment of effluent with metal resistant bacteria

The treatment of the effluent with the metal resistant bacteria were done in Erlenmeyer flasks
containing 150 mL of each samples and 15.0 ± 1.0 mg of cells. The flasks were kept, under
constant agitation, at 30-35 0 C ± 2ºC for 48 hours. After 48 hours, cells were separated from
the medium and residual metal concentrations were determined by ICP-MS

3. Results and Discussion

3.1 Isolation of heavy metal resistant bacteria

Numerous studies have revealed a number of bacterial species which are capable of removing
metals from aqueous environment. In the present study three bacterial species were isolated:
Bacillus spp., Pseudomonas spp. and Staphylococcus spp. from the soil and waste water by
serial dilution and pour plating method using Nutrient agar supplemented with different
heavy metal salts.The biochemical characterstics of the isolated bacteria are given in Table 1.

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 Removal of Heavy Metals from Industrial Effluent Using Bacteria

Table 1: Biochemical Charaterization of isolated bacteria

S.No. Indole MR VP Citrate Urease Nitrate Catalase Oxidase Identified

Utilisation test Reduction bacteria
1 - + - + - + + + Staphylococcus
sp.
2 + + - - + + - - Bacillus sp
3 - - + + - + + + Pseudomonas
sp.

3.2 Measurement of Physico-Chemical parameters of the effluent samples

Samples were assessed for various physicochemical parameters like pH, DO,BOD and COD
and are listed in Table 2. The pH ranged from 6.0-7.9, BOD ranged from 250-480 mg/l, COD
ranged form 490-890 mg/l.

Table 2: Physico-chemical characterstics of the Effluent samples

S.No. Sample pH DO BOD COD

(mg/l) (mg/l) (mg/l)
1. A 6.0 0.2 250 520
2. B 7.2 0.3 360 655
3. C 7.5 0.2 260 542
4. D 6.8 0.4 480 890
5. E 6.2 0.3 380 732
6. F 6.2 0.3 410 764
7. G 7.9 0.4 460 810
8. H 7.1 0.2 220 490
9. I 6.5 0.3 320 632

3.3 Determination of Heavy Metals concentration in industrial Effluent

The concentration of Heavy metals in the effluent samples was determined by ICP-MS and
the metals Cd, Cu, As, Hg and Co were determined in the effluent samples. The
concentration of Cadmium(Cd) ranged from 0.48 to1.62mg/l, concentration of Arsenic (As)
ranged from 0.14 to 0.31mg/l, concentration of Mercury(Hg) ranged from 0.59 to 1.75 mg/l,
concentration of copper (Cu) ranged from 0.21 to 1.35 mg/l, and concentration of cobalt
ranged from 0.08 to 0.91mg/l. Five samples A,C,E,G and H out of nine, were selected for
this study due to high content of heavy metals (Table 3).

Table 3: Heavy metal content in the Effluent samples

S.No. Sample Cd As Hg Cu Co
mg/l mg/l mg/l mg/l mg/l
1. A 0.85 0.31 1.43 0.21 0.60
2. B ND 0.01 0.09 ND 0.02
3. C 1.54 0.14 1.75 1.35 0.91
4. D 0.09 ND ND 0.06 0.05
5. E 0.48 0.22 0.59 0.42 0.24
6. F 0.04 ND 0.02 0.04 0.01
7. G 1.02 ND 1.15 ND 0.08

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 Removal of Heavy Metals from Industrial Effluent Using Bacteria

8. H 1.62 0.18 1.32 0.36 0.32

9. I ND 0.15 ND 0.12 0.06

3.4 Treatment of effluent with metal resistant bacteria

3.4.1 Treatment of metal by Bacillus sp.

The reduction in heavy metal concentration in the effluent sample by Bacillus sp. is indicated
in table 4. The heavy metals Hg and Cu were removed by Bacillus sp. The average Hg
reduction was 45% and Cu reduction was recorded as 62%.

Table 4: Treatment of metal by Bacillus sp.

S.No Sample Hg(before Hg(after Cu(before Cu(after
treatment) treatment) treatment) treatment)
mg/l mg/l mg/l mg/l
1. A 1.43 0.79 0.21 0.08
2 C 1.75 0.88 1.35 0.48
3. E 0.59 0. 33 0.42 0.17
4. G 1.15 0.69 ND ND
5. H 1. 32 0.73 0.36 0.14

3.4.2 Treatment of metal by Pseudomonas sp.

The reduction in heavy metal concentration in the effluent sample by Pseudomonas sp. is
indicated in table 5. The heavy metals Cd, As and Co were removed by Pseudomonas sp. The
average Cd reduction was 56%, average as reduction was 34% and average Co reduction was
recorded as 53%.

Table 5: Treatment of metal by Pseudomonas sp.

S. Samp Cd(bef Cd As(bef As(afte Co(before Co(after
No le ore (after ore r treatment) treatmen
. treatm treatm treatm treatm mg/l t)
ent) ent) ent) ent) mg/l
mg/l mg/l mg/l mg/l
1. A 0.85 0.36 0.31 0.22 0.60 0.29
2 C 1.54 0.62 0.14 0.09 0.91 0.39
3. E 0.48 0.22 0.22 0.15 0.24 0.11
4. G 1.02 0.44 ND ND 0.08 0.05
5. H 1.62 0.78 0.18 0.12 0.32 0.15

3.4.3 Treatment of metal by Staphylococcus sp.

The reduction in heavy metal concentration in the effluent sample by Staphylococcus sp. is
indicated in table 6. The heavy metals Cd and Cu were removed by Staphylococcus sp. The
average Cd reduction was 44% and average Cu reduction was recorded as 34%.

Table 6: Treatment of metal by Staphylococcus sp.

S.No Sample Cd(before Cd (after Cu(before Cu(after
. treatment treatment treatment treatment)
) ) ) mg/l
mg/l mg/l mg/l
1. A 0.85 0.51 0.21 0.14

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 Removal of Heavy Metals from Industrial Effluent Using Bacteria

2 C 1.54 0.82 1.35 0.93

3. E 0.48 0.26 0.42 0.27
4. G 1.02 0.60 ND ND
5. H 1.62 0.86 0.36 0.25

4. Conclusion

From the present study it could be concluded that bacteria play a very important role in the
removal of heavy metals from waste water. In the present study five effluent samples out of
nine were selected to study the removal of heavy metals by bacteria. After treatment it was
found that Pseudomonas sp. and Bacillus sp. were able to remove Cd from the effluent
samples with an average reduction of 56% and 44% respectively. Removal of As was
recorded by Pseudomonas sp. with an average reduction recorded of 34%.Hg was removed
by Bacillus sp. with an average reduction of 45%. Cu was removed by both Bacillus sp and
Staphylococcus sp. with an average reduction recorded of 62% and 34% respectively. Co was
removed by Pseudomonas sp. and the average reduction recorded was 53%.

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