Isolation, Identification and Characterization of Heavy Metal Resistant Bacteria from Industrial Affected Soil in Central India

Abstract

Microorganisms related technologies may provide an alternative or addition to conventional method of metal recovery or metal removal. The present study deals with isolation, identification and characterization of heavy metal resistant bacteria was isolated from industrial affected soils collected from different places at a depth of 0-15 centimeter, in and around Mandideep industrial areas, Raisen district, Central India. Initially, a total of 175 isolates were screened from industrial affected soil. The five isolates were selected based on high level of heavy metal resistances. On the basis of morphology, biochemical revealed that, the isolates were identified as Proteus vulgaris (MR1), Bacillus cereus (MR2), Bacillus decolorationis (MR3), Pseudomonas fluorescence (SS4) and Pseudomonas fluorescence (SS5). The soil isolates showed optimum growth at pH 7.0 and 30°C. The identified isolates were resistant to cadmium (Cd), nickel (Ni), lead (Pb), arsenic (As), and chromium (Cr). The minimal inhibitory concentration (MIC) of soil isolates against Cd, Cr, Ni, Pb and As was determined in solid media. The identified heavy metal resistant bacteria could be effective and useful for the bioremediation of heavy metal contaminated soil.

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Ahirwar et al Int. J. Pure App. Biosci. 4 (6): 88-93 (2016) ISSN: 2320 – 7051
Copyright © December, 2016; IJPAB 88
Isolation, Identification and Characterization of Heavy Metal Resistant
Bacteria from Industrial Affected Soil in Central India
Narendra Kumar Ahirwar1, Govind Gupta2, Ravindra Singh1, Vinod Singh2*
1Mahatma Gandhi Chitrakoot Gramodaya Vishwavidhyalaya, Chitrakoot, (M.P.), India
2Department of Microbiology, Barkatullah University, Bhopal, (M.P.), India
*Corresponding Author E-mail: vsingh3@rediffmail.com
Received: 20.12.2016 | Revised: 28.12.2016 | Accepted: 31.12.2016
INTRODUCTION
Rapid increase of population and the increased
demand for industrial establishments to meet
human needs have created problems such as
over exploitation of available resources,
increased pollution taking place on land, air
and water environment. Heavy metal pollution
of soil and wastewater is a significant
environmental problem1. Wastewaters from
the industries and sewage sludge applications
have permanent toxic effects to human and the
environment2. According to EPA3,
bioremediation is defined as a managed of
spontaneous process in which microbiological
process are used to degrade, break down or
transform hazardous contaminants to less toxic
or nontoxic forms, thereby remedying or
removing and eliminating contaminants from
environment media.
Available online at www.ijpab.com
DOI: http://dx.doi.org/10.18782/2320-7051.2424
ISSN: 2320 – 7051
Int. J. Pure App. Biosci. 4 (6): 88-93 (2016)
ABSTRACT
Microorganisms related technologies may provide an alternative or addition to conventional
method of metal recovery or metal removal. The present study deals with isolation, identification
and characterization of heavy metal resistant bacteria was isolated from industrial affected soils
collected from different places at a depth of 0-15 centimeter, in and around Mandideep industrial
areas, Raisen district, Central India. Initially, a total of 175 isolates were screened from
industrial affected soil. The five isolates were selected based on high level of heavy metal
resistances. On the basis of morphology, biochemical revealed that, the isolates were identified
as Proteus vulgaris (MR1), Bacillus cereus (MR2), Bacillus decolorationis (MR3), Pseudomonas
fluorescence (SS4) and Pseudomonas fluorescence (SS5). The soil isolates showed optimum
growth at pH 7.0 and 30°C. The identified isolates were resistant to cadmium (Cd), nickel (Ni),
lead (Pb), arsenic (As), and chromium (Cr). The minimal inhibitory concentration (MIC) of soil
isolates against Cd, Cr, Ni, Pb and As was determined in solid media. The identified heavy metal
resistant bacteria could be effective and useful for the bioremediation of heavy metal
contaminated soil.
Key words: Contaminated Soil, Heavy metals, Soil Bacteria, and Bioremediation.
Research Article
Cite this article: Ahirwar, N.K., Gupta, G., Singh, R. and Singh, V., Isolation, Identification and
Characterization of Heavy Metal Resistant Bacteria from Industrial Affected Soil in Central India, Int. J.
Pure App. Biosci. 4(6): 88-93 (2016). doi: http://dx.doi.org/10.18782/2320-7051.2424

Ahirwar et al Int. J. Pure App. Biosci. 4 (6): 88-93 (2016) ISSN: 2320 – 7051
Copyright © December, 2016; IJPAB 89
Microorganisms used chemical contaminants
as an energy source using their metabolic
process throughout the microbiological
process. However, the excessive amounts
inorganic nutrients in soil cause microbial
inhibition4. The microorganisms in particular
have the abilities to degrade, detoxify and even
accumulate the harmful organic as well as
inorganic compounds.
There are different sources of heavy
metals in the environment such as: natural,
agricultural, industrial, domestic effluent,
atmospheric sources and other sources.
Activities such as mining, electroplating,
metallurgical, smelting operations and
agriculture have contaminated extensive areas
of world such as Japan, Indonesia and China
mostly by heavy metals such as Cd, Cu and
Zn5. Heavy metal (HM) pollution is a major
environmental problem6, that reduces crop
production and food quality. Unlike organic
contaminants, metals are not degradable and
thus remain in the environment for long
periods of time; when present at high
concentrations, metals can negatively affect
plant metabolism7. In our previous studies, we
have studied the feasibility of remediation of
soils contaminated with Cr using different
floriculture plant species, i.e., tuberose
(Polianthes tuberosa), chrysanthemum,
calendula, aster and dahlia8-9. From these
studies, it was found that majority of the plant
species could tolerate at the most 10–15 mg
Cr/kg soil. However, the contaminated sites
would have very high levels of Cr and at times
would even be unfit for cultivation of the
crops.
During the last few decades, the
Mandideep, State Madhya Pradesh, Central
India, has undergone rapid social and
economic development. It could face public
health and ecological problems if heavy metal
loads exceed a critical value. Little
information is available on heavy metal
concentrations on soils of Mandideep10. There
is a need for innovative treatment technologies
for the removal of heavy metal ions from soil,
water bodies and wastewater. Different
microbes have been proposed to be efficient
and economical alternative in removal of
heavy metals from soil and water11. The
objective of this study is to determine heavy
metals resistance of bacteria, MIC, growth
studies, and biochemical characteristics were
used to exploit these isolates for cleanup of
industrial affected soil, wastewater and
sewage.
MATERIALS AND METHODS
Soil Collection
Soil samples were collected randomly from
industrial contaminated soil areas nearby
pharmaceutical, textile, food and beverages,
leather, steel and iron workshop, graphite, and
tractor manufacturing industry of Mandideep
District Raisen of Madhya Pradesh, Central
India. Soil samples were collected in sterilized
polythene bags and immediately bought to the
Microbiology laboratory, Department of
Microbiology Barkatullah University Bhopal,
Madhya Pradesh, for bacteriological analysis.
Isolation of bacteria
The microbial strains were isolated from the
collected soil samples by serial dilution
technique. Selective isolation of bacterial spp.
was performed by spreading the samples on
their individual media.
Identification and characterization of
bacteria from soil sample
Individual distinct colonies were further
undergone repeated sub-culturing. Selected
colonies were grown on nutrient agar media
(Himedia, India). Identification of microbes by
using morphological, cultural and biochemical
characteristics for the activities of motility,
oxidase, catalase, VP test, MR test, starch and
gelatin hydrolysis, indole production and
citrate utilization according to Bergey’s
manual of systemic bacteriology12 .
Determination of optimal bacterial growth
conditions
The optimal growth conditions with reference
to temperature and pH were determined. The
isolates were grown in nutrient agar medium
with different temperature between 250C,
300C, 370C and 450C and incubation was
carried out at pH values between (6.0, 7.0, 7.5,
and 8.0). The optical density of the log phase

Ahirwar et al Int. J. Pure App. Biosci. 4 (6): 88-93 (2016) ISSN: 2320 – 7051
Copyright © December, 2016; IJPAB 90
growing cultures (12-14 h) conditions was
noted at 600 nm using spectrophotometer to
determine the growth.
Determination of Minimum Inhibitory
Concentration (MIC)
The stock solutions of K2Cr2O7, CdCl2, Pb
(NO3)2, ZnSO4.7H2O NiCl2, and HgCl2 were
prepared in sterile deionized water and
sterilized by autoclaving at 1210C for 15 min.
The culture grow at a given concentration
were subsequently transferred to the next
concentration. Based on the evaluation,
minimum inhibitory concentration (MIC) was
determined at 300C for 6 days. Five bacterial
strains showing higher MIC values for Cr, Cd,
Pb, Zn, Ni, and Hg were selected for further
studies.
Evaluation of Heavy Metal Tolerance
Tolerance of selected bacterial strains to
various heavy metals was determined by agar
dilution method13. Freshly grown agar cultures
of these isolates were inoculated aseptically on
nutrient agar plates supplemented individually
with other heavy metals. The metal salts used
were K2Cr2O7, Pb (NO3)2, (Merck),
ZnSO4.7H2O, CdCl2, NiCl2 and HgCl2
(Qualigens). The metal ion concentration
tested ranged from 10 to 150 mg/ L. Plates
were incubated aerobically at different
temperature between 250C, 300C, 370C and
450C for 6 days and observed for growth.
RESULTS
Isolation of heavy metal resistant bacteria
In the present study we identify and
characterize heavy metals resistant bacteria
isolated from industrial affected soil. 150
colonies were screened from initial level of
heavy metal supplemented nutrient agar
medium. 25 isolates were selected in the
secondary screening from soil. Finally five
strains were selected based on high degree of
heavy metals resistances were used for further
studies. The strains Proteus vulgaris (MR1),
Pseudomonas fluorescence (SS4) and
Pseudomonas fluorescence (SS5) was Gram-
negative, rod shaped motile and Bacillus
cereus (MR2), Bacillus decolorationis (MR3)
was Gram-positive, rod shaped motile
bacteria. The sewage isolates showed optimum
growth at 30°C and pH 7.0. The biochemical
characteristics of soil bacteria were shown in
(Table- 1, 2 and 3).
Growth studies of bacteria and
simultaneously resistant to heavy metals
Growth studies of MR1, MR2, MR3, SS4 and
SS5 were carried out in nutrient agar medium
with Cr, Cd, Pb, As, Ni (200 mg/l) and Hg
(100 mg/l) were prepared from stock solutions.
The measurements from the cultures incubated
for 48 h were in good agreement according to
bacterial resistance for each heavy metal. All
isolates were found to be sensitive to for Cr
and Hg. Thus, microorganisms having
combined abilities of simultaneous resistance
to heavy metals and their biotransformation to
non-toxic form will be potentially useful for
detoxification of heavy metals polluted soil.
MIC of heavy metal
Soil bacteria MR1, MR2, MR3, SS4 and SS5
showed high degree of resistance to all heavy
metals, MIC values varying concentration
from 0.35 - 17.5 mM. Among the heavy
metals zinc and nickel was less toxic, where as
chromium and mercury were highly toxic to
all strains. MIC of heavy metal were shown in
(Table- 4 & Fig 1).
Table: 1 Morphological characterization of isolated bacterial species
S. No.
Morphological
characteristics
Isolated bacterial colonies
MR1
MR2
MR3
SS4
SS5
1
Colour of the colony
White
White
White
White
White
2
Shape of the cell
Rod
Rod
Rod
Rod
Rod
3
Gram s Staining
Negative
Negative
Positive
Positive
Positive
4
Motility
Motile
Motile
Motile
Motile
Motile
Note: MR1 -P. vulgaris, MR2- B. cereus, MR3 -B.decolorationis, SS4- P. fluorescence, SS5- P. fluorescence

Ahirwar et al Int. J. Pure App. Biosci. 4 (6): 88-93 (2016) ISSN: 2320 – 7051
Copyright © December, 2016; IJPAB 91
Table: 2 Biochemical characterization of isolated bacterial species
S. No.
Morphological
characteristics
Isolated bacterial colonies
MR1
MR2
MR3
SS4
SS5
1
Indole
Positive
Negative
Negative
Negative
Negative
2
Methyl red
Positive
Negative
Negative
Negative
Negative
3
Voges-proskauer test
Negative
Negative
Positive
Negative
Negative
4
Citrate utilization test
Positive
Positive
Positive
Negative
Negative
5
Urease hydrolysis
Variable
Variable
Negative
Negative
Negative
6
Oxidase
Negative
Positive
Negative
Positive
Positive
7
Catalase
Positive
Positive
Positive
Positive
Positive
Note: MR1 -P. vulgaris, MR2- B. cereus, MR3 -B.decolorationis, SS4- P. fluorescence,
SS5- P. fluorescence
Table: 3 Bacterial growth at different temperature
S. No.
Temperature (˚C)
Isolated bacterial colonies
MR1
MR2
MR3
SS4
SS5
1
4
-
-
-
-
-
2
25
-
+
+
+
+
3
30
+
+
+
+
+
4
37
+
+
+
+
+
5
45
-
-
-
+
+
Note: + positive, - negative
Table: 4- MIC of heavy metals against soil bacteria
S. No.
Heavy metals
Isolated bacterial colonies
MR1
MR2
MR3
SS4
SS5
1
Chromium
5.0
6.5
13
5.9
5.8
2
Cadmium
5.1
7.0
4.8
7.3
7.3
3
Lead
6.2
4.6
3.7
8.2
6.1
4
Zn
10.0
6.0
5.4
17.5
16.8
5
Nickel
8.8
0.79
0.35
13.2
12.5
6
Mercury
0.12
1.3
0.0
5.4
5.2
Note: Heavy metal concentration in mM.
Fig. 1: Graphical representation of heavy metals resistance by isolated bacterial strains
0
2
4
6
8
10
12
14
16
18
20
Cr Cd Pb Zn Ni Hg
Heavy metals conc. in mM
MIC conc. of heavy metals by bacterial strains
MR1
MR2
MR3
MR4
MR5

Ahirwar et al Int. J. Pure App. Biosci. 4 (6): 88-93 (2016) ISSN: 2320 – 7051
Copyright © December, 2016; IJPAB 92
CONCLUSION
The bioremediation process is influenced by
various factors such as soil type, pH,
temperature, nutrient, amendments and
oxygen. The ability of microbial stains to grow
in the presence of heavy metals would be
helpful in the contaminated soil treatment
where microorganisms are directly involved in
the decomposition of organic matter in
biological processes for soil treatment. In the
present study high degree of heavy metals
resistance associated with multiple heavy
metals was detected in industrial affected soil
bacteria. In the present study the order of
resistance of Cr was: MR3(13mM)
>MR2(6.5mM) >SS4 (5.9mM) >SS5(5.8mM)
>MR1(5.0mM), resistance of Cd was:
SS5(7.3mM) = SS4 (7.3mM) >MR2(7.0mM)
>MR1(5.1mM) >MR3(4.8mM), resistance of
Pb was: SS4(8.2mM) >MR1(6.2mM)
>SS5(6.1mM) >MR2(4.6mM)
>MR3(3.7mM), resistance of Zn was:
SS4(17.5mM) >SS5(16.8mM)
>MR1(10.0mM) >MR2(6.0mM)
>MR3(5.4mM), resistance of Ni was:
SS4(13.2mM) > SS5(12.5mM) >
MR1(8.8mM) >MR2(0.79mM)
>MR3(0.35mM) and the order of resistance of
Hg was: SS4(5.4mM) > SS5(5.2mM) >
MR2(1.3mM) > MR1(0.12mM)
>MR3(0.0mM). Among the heavy metals zinc
and nickel was less toxic, where as chromium
and mercury were highly toxic to all bacterial
isolates. The efforts must be directed towards
faster and economic restoration of heavy
metals and pesticide contaminated soil to
secure the soil quality and food and health of
mankind. The future research should
investigate the heavy metals and pesticide
bioremediation potential of recombinant as
well as indigenous bacteria with and without
supplements.
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