Bioremediation of Pb and Cd by some soil bacteria in Basra- Iraq

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I
Summary
Assessment of lead (Pb) and cadmium (Cd) content in soil samples from
Fao, Um-Qasr and Al-Zubair districts of Basra city southern Iraq were
undertaken. Top soil samples (0-20 cm) were taken from these districts during
the study period (January 2013). Total metal content is important because it
determines the size of the metal pool in the soil and thus the potential for metal
up take, therefore, soil samples were analyzed for total and bioavailability of
lead and cadmium. Soil textural classification showed that, there were two
types of soil textures (silt loam and loam sand) on the basis of percentage of
sand, silt and clay for division of soils into textural classes. The pH values of the
soils in all the sites ranged from 8.27to 8.56 with a mean value of 8.39 showing
that the soils were alkaline in nature. The electrical conductivity differs
according to the district and was lowest in Al-Zubair (2.3ms/cm), while the
highest was found in Fao and Um-Qasr soils which reached 10.5 and 9.84
ms/cm respectively. Total organic carbon (%) content ranged from 0.1 to 0.5
with a mean value of 0.26, which indicated signifying presence of degradable
substances and increased microbial activities in the soil. The total concentration
of Pb and Cd had different values with different soils, the higher value recorded
in Um-Qasr 28.39 and 6.81μg/g, followed by Fao 19.80 and 5.96 μg/g,
respectively, while the lowest concentrations were occurred in Al-Zubair 14.16
and 4.30 μg/g respectively. The results of bioavailability showed that Cd was
more available than Pb in all districts.
Bacteria were isolated from soil samples, purified and identified using their
biochemical profile and 16S rRNA sequences as Deinococcus radiodurans,
Shewanella oneidensis and Bacillus thuringiensis. The ability of these bacteria to
tolerate metal concentrations was explored by determining their minimum
inhibitory concentrations for Pb and Cd. B. thuringiensis exhibited lower MIC
for Cd (50 mg/l), and higher MIC for Pb (1800 mg/l). S. oneidensis showed

II
higher MIC for Cd (1000 mg/l), and its MIC for Pb was (700 mg/l). While the
MIC of D. radiodurans for Cd and Pb were 600 and 400 mg/l respectively.
The bioremediation ability of isolating bacteria has been studied through two
routes, including bioaccumulation and biosorption at pH 6.5, 30°C and speed
shaking of 180 rpm.
The present study showed that the accumulation of these metals by D.
radiodurans was gradual and the amount increased in direct proportion to initial
metal concentration up to an extent that ranged from 10 - 100 mg/g biomass. The
maximum uptake of Pb and Cd were obtained at initial concentrations of 50and
100 mg/l, respectively, and the values were 0.33 and 6.84 mg/g. Contact duration
increased the amount of metal bioconcentrated by this bacterium at 50 and 100
mg/l maximum uptake of Pb and Cd, were at 6 and 24h of exposure
respectively.
For S. oneidensis maximum uptake of Pb and Cd were obtained at an initial
concentration 50and 100 mg/l, respectively, and the values were 3.98 and 26.77
mg/g. Contact duration increased the amount of metal bioconcentrated by this
bacterium at 50 and 100 mg/l tested concentrations maximum uptake of Pb and
Cd, were at 48h of exposure time.
B. thuringiensis took the same manner of accumulation, where it accumulates
high amount of Pb and Cd at 11.95 and 23.2 mg/g in concentration 50 and 100
mg/l respectively at time 24 and 6 h respectively.
The accumulation of heavy metals by the cells of study bacteria was
demonstrated by TEM, which indicated presence of metals within the cell
membrane and inside of cells in addition to some changes in the cells shape and
size in response to heavy metals exposure and sporulation in case of B.
thuringiensis.
The study bacteria were used to test their ability in remediation Pb and Cd by
biosorption process; the effects of various parameters such as contact time, metal
concentration were examined.

III
Optimum removal (%) of Pb and Cd by D. radiodurans was found to be
63.46 and 31.23 respectively at concentration 50 mg/l at 2h of the incubation
period.
By using S. oneidensis the maximum removal ( %) of Pb was 51.06 at
concentration 50mg/l at 2h, while for Cd was 42.64 in concentration 5mg/l at 2h.
B. thuringiensis recorded highest removal (%) of Pb and Cd which was 69.64
and 93.06 in concentration 50mg/l at 2h.
The physical and chemical characteristics of biosorbents are important for
understanding the metal binding mechanism on the biomass surface, therefore the
FTIR and XRD were used to demonstrate the adsorption of heavy metals to cells,
through analyzing the number and position of the functional groups available for
the binding of heavy metals ions and determined the crystallographic nature of
participate which showed many functional groups in bacterial cell wall
responsible for such biosorption and there's nanoparticles form from participate
these metals on bacterial cell wall .

IV
pageTitle
No. 1-18Chapter one: Introduction and literatures review
1-3Introduction1.1
3The aim1.2
4-18Literatures review1.3
4-5Soil pollution with heavy metals1.3.1.
5-6Bioavailability of heavy metals1.3.2.
7-8Technologies for the remediation of polluted soils1.3.3.
8-9Bioremediation1.3.4.
9-10Metals contamination and microorganisms1.3.5.
10-11Bioremediation of Metals by Microbial Processes1.3.6.
12-18Examples of bacteria used in bioremediation of
heavy metals
1.3.7.
12-13Deinococcus radiodurans1.3.7.1.
Table of contents

V
13-14Application of D. radiodurans in bioremediation1.3.7. 1.1.
15Shewanella oneindensis1.3.7.2.
15-16Application of Shewanella oneindensis in
bioremediation
1.3.7.2.1.
16-17sthuringiensiacillusB1.3.7. 3.
17-18Application of Bacillus thuringiensis in
bioremediation
1.3.7. 3.1.
19-38Chapter two: Materials and methods
19-38Materials and methods2
19-26Materials2.1
19-21Chemicals2.1.1.
21-22Equipments2.1.2.
23Media and media components2.1.3.
24Isolation media2.1.4.
24-25Biochemical Tests Media2.1. 5.
25-26Reagents2.1.6.

VI
27-38Methods2.2.
27The study aria2.2.1.
29Soil sampling and handling2.2.2.
29-30Soil analyses2.2.3.
29Soil texture2.2.3.1
29PH2.2.3.2.
29Electrical conductivity (EC)2.2.3.3.
29Total organic carbon (Organic C)2.2.3.4 .
30Total concentration of heavy metals in soils2.2.3.5.
30Heavy metals Bioavailability2.2.3.6.
31Isolation of bacteria2.2.4.
32Identification of bacteria2.2.5.
33-35Molecular identification2.2.5.1.
33Total genomic DNA extraction2.2.5.1.1.

VII
3416s rRNA2.2.5.1.2.
34PCR products purification2.2.5.1.3.
35Bioinformatics analysis of bacteria samples2.2.5.1.4.
35Preparation of heavy metals concentrations2.2.6.
35Determination of minimum inhibitory
concentrations (MIC) of heavy metals.
2.2.7.
35Bioaccumulation of heavy metals by Bacteria.2.2.8.
36Digestion of bacterial biomass for atomic absorption
measurement for atomic absorption measurement.
2.2.8.1.
36Biosorption of heavy metals by Bacteria2.2.9.
37-38Characterization study2.2.10.
37Transmission electron microscope2.2.10. 1.
38FTIR analysis2.2.10.2.
38X-ray powder diffraction analysis (XRD)2.2.10.3.
38Statically analysis2.2.11.
39-72Chapter three: Results

VIII
39-72Results3
39-41Physical and chemical properties3.1.
39Soil texture3.1.1.
39-40PH, Electrical Conductivity and Total organic
carbon
3.1.2.
40Total concentration of heavy metals3.2.
41Bioavailability of heavy metals3.3.
41-52Isolation and Identification of bacteria3.4.
41-43Deinococcus radiodurans3.4.1.
41-43Cultivation characteristic, microscopic examination
and biochemical tests
3.4.1.1.
44-46Shewanella oneidensis.3.4.2.
3.4.2.1.
46-48Bacillus thuringiensis3.4.3.
3.4.3.1.
49-52Molecular identification of bacteria3.4.4.

IX
52-53Minimum inhibitory concentration (MIC)3.5
54-57Bioaccumulation study3.6.
55-56Shewanella oneidensis3.6.2.
56-57Bacillus thuringiensis3.6.3.
58-61Biosorption study3.7.
59-60Shewanella oneidensis3.7.2.
61Bacillus thuringiensis3.7.3.
62-72Characterization study3.8.
62-65FTIR analysis3.8.1.
62-63Bacillus thuringiensis3.8.1.1.
64Deinococcus radiodurans3.8.1. 2.
65Shewanella oneidensis3.8.1.3.

X
66-69X-ray powder diffraction analysis (XRD)
69Shewanella oneidensis3.8.3.
69-72Transmission electron microscope3.8.3.
70-71Shewanella oneidensis3.8.3.2.
73-98Chapter four: Discussion
73-77Physiochemical parameters4.1
77-79Isolation and identification of bacteria4.2
78Shewanella oneidensis4.2.2.
78-79Bacillus thuringiensis.4.2.3.

XI
79-82Minimum inhibitory concentration (MIC)4.3.
82-85Bioaccumulation study4.4.
86-90Biosorption study4.5.
91-98Characterization study4.6.
91-94The Fourier transform infrared (FTIR).4.6.1.
94-96X-ray powder diffraction analysis (XRD)4.6.2.
97-98Transmission electron microscope (TEM)4.7.
99-100Conclusions and recommendations
99Conclusions5.1.
100Recommendations5.2.
101-127References

XII
PageTitleTable
19-21All chemicals used in the study1
21-22All Equipments used in the study2
23Media and media components3
39Soil texture of sample studied.4
40pH, electrical conductivity (mS/cm) and Total
organic carbon (%) in sample soils.
5
40Total concentration of Pb and Cd (μg/g) in soil
samples.
6
41Bioavailability (μg/g) of Pb and Cd in soil
samples.
7
43Biochemical characteristics of D. radiodurans
isolate from soil.
8
46Biochemical characteristics of S. oneidensis isolate
from soils.
9
48Biochemical characteristics of B. thuringiensis
isolated from soils.
10
52Minimum inhibitory concentration of Cd and Pb
by D. radiodurans, S. oneidensis and B.
thuringiensis.
11
59Biosorption (%) of Lead and Cadmium at
different period of incubations and different
concentrations by D. radiodurans.
12
60Biosorption (%) of Pb and Cd at different
incubation period and different concentrations by
13
List of tables

XIII
S. oneidensis.
61Biosorption (%) of Pb and Cd at different
incubation period and different concentrations by
B. thuringiensis.
14
63Assignments of Infrared absorption bands15
PageTitleFigure
28A map shows the sites of sampling1
50PCR products (A) M, Fermentas GeneRuler 1000 bp;
Lanes: 1, PCR products from samples D; 2, PCR products
from samples S.; N, negative control.(B) M, Fermentas
GenRuler 1000pb DNA ladder Plus; Lanes 1, PCR products
from sample B., N, negative control.
2
54Bioaccumulation of Pb by D. radiodurans at different
incubation period and different concentrations.
3
55Bioaccumulation of Cd by D. radiodurans at different
incubation period and different concentration
4
56Bioaccumulation of Pb by S. oneidensis during different
5
56Bioaccumulation of Cd by S.oneidensis during different
6
57Bioaccumulation of Pb by B. thuringiensis during different
incubation periods and different concentrations.
7
List of figures

XIV
57Bioaccumulation of Cd by B. thuringiensis during different
8
63The FTIR Spectra of B. thuringiensis with Pb (II), Cd (II)
loaded and unloaded.
9
64The FTIR Spectra of D. radiodurans with Pb (II), Cd (II)
lodaded and un loaded.
10
65The FTIR Spectra of S. oneidensis with Pb (II), Cd (II)
lodaded and un loaded.
11
67XRD analysis of B. thuringiensis biomass before and after
Pb and Cd biosorption.
12
68XRD analysis of D. radiodurans biomass before and after
Pb and Cd biosorption.
13
69XRD analysis of S. oneidensis biomass before and after Pb
and Cd biosorption.
14
70Transmission electron micrographs of D. radiodurans, a:
control, b: treated with 50mg/l Pb, c: Treated with50 mg/l
Cd (Scale bar 0.5μ).
15
71Transmission electron micrographs of S. oneidensis, a:
control, b: treated with 50 mg/l of Cd for 24h, c: Treated
with 50mg/l of Pb for 24h (Scale of bar 0.5μ and 2 μ.).
16
72Transmission electron micrographs of B. thuringiensis, a:
control, b: treated with 50mg/l Cd, c:Treated with 50 mg/l
Pb (Scale of bar 0.5μ).
17

XV
PageTitlePicture
42Colony morphological of D. radiodurans grows on
growth medium TGY.
1
42Microscopic shape of D. radiodurans (gram stains)
(1000X). Bacteria appear gram positive cocci and four
cell form tetrad.
2
43D. radiodurans as it appears under FE-SEM with cell
dimension measurement.
3
44Colony morphological of S. oneidensis grows on the
isolate medium MB.
4
45Microscopic shape of S. oneidensis (gram stains)
(1000x).Bacteria appear gram negative curve rode.
5
45S. oneidensis as it appears under FE-SEM, with cell
6
47Colony morphological of B. thuringiensis grow on L.B.
medium.
7
47Microscopic shape of B. thuringiensis (gram stains)
(1000X). Bacteria appear gram positive bacilli with
terminal spore.
8
48B. thuringiensis as it appears under FESEM, with cell
dimension measurements.
9
53MIC experiment A and B, represent the sensitivity of
the bacteria to Pb and Cd. C and D represent resistance
of the bacteria to Pb and Cd.
10
List of Pictures

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1
Introduction and literatures review
1.1- Introduction
Pollution is known as the presence of pollutant in the environment while
pollutant refers to a substance, organism or energy form present in amounts that
impair or threaten an ecosystem to the extent that its current or future uses are
precluded. Nowadays, heavy metals pollution considered is one of the most
important environmental concerns.
Anthropogenic activities like metalliferous mining and smelting, agriculture,
waste disposal or industrial discharge, war, and nuclear processes are
responsible for pollution by a variety of heavy metals such as Ag, As, Au, Cd,
Co, Cr, Cu, Hg, Ni, Pb, Pd, Pt, Rd, Sn, Th, U and Zn, (Jain et al., 2012).
Heavy metals have a major problem to human health and environmental issues
due to the high incidence as a contaminant, low solubility in biota and
classification of various heavy metals as carcinogens and mutagens (Rani et al.,
2010). Heavy metals can produce harmful effects on human health when they
are taken up in amounts that cannot be processed by the organism. In addition,
these metals cannot be degraded to harmless products and hence persist in the
environment indefinitely.
Metal wastes could be reached into the human and animals through their
inhalation, consumption of contaminated food, water, and skin contact. The
exposure to these metals for long periods may lead to the permanent damage of
organelles. For these reasons several methods have been designed for the
treatment and removal of heavy metals in contaminated site (Akhtar et al.,
2013). Physico-chemical methods have been used, such as electrochemical
treatment, ion exchange, precipitation, reverse osmosis, evaporation, and
sorption (Congeevaram et al., 2007). But these methods have disadvantages,
including economically expensive, incomplete metal removal, requirements
higher reagent energy, and generation of toxic sludge. In some cases it may

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change the environment properties and spread contaminants from one to another
would also increase the consumption of non renewable resources (Chojnacka,
2010). In this endeavor biological methods have the particular effect for the
accomplishment of this goal and they are economical.
Bioremediation is a natural process which depends on bacteria, fungi, and
plants to change pollutants as these organisms carry out their normal life
functions. These organisms have the ability of using chemical contaminants as
an energy source in their metabolic processes. Thus, bioremediation affords
substitute tool to destroy or reduce the harmful contaminants through
biological activity and this method has an effective cost (Salem et al., 2012).
Bioaccumulation is the active method of metal accumulation by living cells.
The capacity of living cells to remove metal ions from environment is
influenced by environmental growth conditions, as temperature, pH, and
biomass concentrations (Abd El-Raheem et al., 2013). For living cells, metal
uptake is also facilitated by the production of metal binding proteins also called
as metallothioneins (MTs) or low molecular weight cysteine-rich proteins.
Biosorption can be defined as the removal of a metal or metalloid species,
compounds and particulates from environment independently of biological
material (El-Meleigy et al., 2011). Large amounts of metals can be accumulated
by a variety of processes independent on metabolism. Biosorption is a property
of both living and dead organisms (and their components) and has been
heralded as a promising biotechnology for pollutant removal from solution,
and/or pollutant recovery, for a number of years, because of its efficiency,
simplicity, analogous operation to conventional ion exchange technology, and
availability of biomass (Gadd, 2009). Biosorbents possess metal-sequestering
property can be used to decrease the concentration of heavy metal ions in
solution from ppm to ppb level. It can effectively sequester dissolved metal ions
out of dilute complex solutions with high efficiency and quickly; therefore it is

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an ideal candidate for the treatment of high volume and low concentration of
complex heavy metal wastes (Wang and Chen, 2006).
The Biosorbents behavior of metallic ions is a function of the chemical
make-up of the microbial cells of which it consists (Costley and Wallis, 2001).
Mechanisms responsible for biosorption, although understood to a limited
extent, may be one or a combination of ion exchange, complexation,
coordination, adsorption, electrostatic interaction, chelation and micro
precipitation (Vijayaraghavan and Yun, 2008; Wang and Chen, 2006).
1.2. The aim of the study
1- Use soil sample to Isolation three bacterial species Bacillus thuringiensis;
Deinococcus radiodurans and Shewanella oneidensis and identification
these bacteria by using biochemical and molecular tools.
2- Study their ability to tolerate heavy metals.
3- Study the bioremediation ability of these bacteria to heavy metals through
both bioaccumulation and biosorption.
4- To complete the study, an attempt had been made to determine the
functional groups of bacterial surface using Fourier transform
infrared (FTIR), and X-ray powder diffraction analysis (XRD) had been
used to determine the crystallographic structure and chemical composition
of metal bound to the biosorbents. In addition to the use of transmission
electron microscope for detection the position of metals take up by bacteria
through active up take.

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1.3. Literatures review
1.3.1. Soil pollution with heavy metals
Soil is a non-renewable resource essential to civilization (Emili, 2011),
and it plays a vital role in completing the cycling of major elements required by
biological systems, transforming and recycling sunlight, storing energy and
matter through plants and animals, decomposing organic wastes, detoxifying
certain hazardous compounds and a medium for plant growth by supplying
physical support, water, essential nutrients, and oxygen for roots, providing
human food and fibre needs (Caporali, 2004). The resource “soil” is a
meaningful crossroads between the different components and processes of
terrestrial ecosystems. It is necessary to the proper functioning of an ecosystem,
contributing to the system’s ability to withstand the adverse effects of such
disturbances as drought, pests, pollution, and human exploitation, including
agriculture (Gregorich et al., 1996).
Soils are presently being degraded through salinization, erosion, sealing,
pollution, loss of organic matter and biodiversity, leading to the deterioration of
the soil’s physical, chemical and biological properties worldwide.
Metal pollution in soils has become one of the most serious environmental
problems of worldwide concern, because of their widespread use and
distribution, and particularly their toxicity to human beings and the biosphere
(Alkorta et al., 2004). Heavy metals and metalloids enter in the ecosystem
through both natural and anthropogenic processes. Despite some soils have been
found to have a high background of some trace elements, toxic to plants and
wildlife due to extremely high concentrations of these elements in the parent
materials (Violante et al., 2010), but heavy metals are present in soils above all
as a result of human activities, such as the burning of fossil fuels, mining and
smelting of metalliferous ores, municipal wastes, agricultural activities, means
of fertilizers, pesticides, long term application of urban sewage sludge's,

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industrial activities such as waste disposal, waste incineration and vehicle
exhausts (Garbisu and Alkorta, 2001 and Alkorta et al., 2004). Metals in soils
have frequently been reported to have a negative effect on soil biological
parameters such as, soil microbial biomass, composition and diversity of the
biomass, mineralization of organic matter, different microbial processes, soil
enzyme activities with concomitant negative effects on soil fertility and
functioning (Leita et al., 1995; Kelly and Tate, 1998 and Giller et al., 1998).
There are many local studies in Iraq picked up contaminated of soil with
heavy metals at different cities. Hussein et al. (2012) studied concentrations of
heavy metals in the soils beside the Euphrates river in Thi-Qar city and the
result showed that the concentration of heavy metals increased as Cd Cr Pb
Ni Cu Zn Fe. Sultan et al., (2012) recorded that, the concentration of
heavy metals in soil differs from district to another. Khwedim et al. (2009)
studied the distribution of some heavy metals in the soil of Basra city south of
Iraq, and they founded that the average of heavy metals concentration were 36,
20, 14, 4.9, 3, 1358 (ppm) for Pb, Ni, Co, Cd, Cr, and Fe respectively.
1.3.2. Bioavailability of Heavy Metals
The definition of the bioavailability is “the degree to which chemicals
present in the soil may be absorbed or metabolized by human or ecological
receptors or are available for interaction with biological systems” (ISO, 2005).
The bioavailability, depending on a specific target organism and specific
contaminants, includes also the following aspects: exposure time, transfer of
contaminants from soil to organisms, their accumulation in the target organisms,
and the subsequent effects (Violante et al., 2010). Factors like pH, organic
matter, and electrical conductivity should be considered because they can
modify the bioavailability. The soil characteristic among other influences the
speciation of the metals, which leads to higher or lower viable fraction and too
long or short residence time of heavy metals in soil (Kalis, 2006; Feng et al.,

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2005and Epelde et al., 2009). To estimate the bioavailability of heavy metals
and thereby predict their impact on the soil ecosystem, no single method is
recognized universally (Soriano-Disla et al., 2010). In general, there are two (1)
by chemical methods (e.g., Extraction methods) which quantify a defined
available fraction of a well defined class of contaminants assumed to be
available for specific receptors (e.g., Macro and Meso fauna living in soil, (2)
by biological method which expose organisms to soil or soil elutes to predict the
amount of contaminants taken up by the organisms and to monitor effects
(Harmsen, 2007).
The total metal content of the soil is not a good indicator for soil metal
contamination and to the availability of metals to organisms; therefore, there
are many studies handled and studied the bioavailability of heavy metals in the
soil to determine whether or not the soil is polluted with heavy metals.
Kaličanin et al., (2014) studied the bioavailability of Pb, Cd, and Cu from the
soil, which is available for absorption by plants, and analysis of soil was done
by fractions. Wang et al., (2010) used a modified sequential extraction method
to investigate the distribution and speciation of Cd, Cu, Pb, Fe and Mn in the
shallow sediments of Jinzhou Bay, Northeast China. This site was heavily
contaminated by nonferrous smelting activities. Siebielec et al. (2006) studied
metal availability by using, neutral salt extractions, sequential extraction and an
in vitro test for Pb bioaccessibility. The study demonstrated the relatively low
availability of metals in long-term contaminated soils. Yuan et al. (2004)
applied BCR-sequential extraction protocol to obtain metal distribution patterns
in marine sediments from the East China Sea. The results showed that both the
total contents and the most dangerous non-residual fractions of Cd and Pb were
extremely high.

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1.3.3. Technologies for the remediation of polluted soils
The cleaning of contaminated soils from heavy metals is the most difficult
task, particularly on a large scale due to the dynamic nature of metal
interactions in soils (Hietala and Roane, 2009). Compared to organic pollutants,
the toxic metals cannot be mineralized / degraded and then their residence time
in the soil is of the order of thousands of years. Unfortunately, traditional
management of soils polluted with heavy metals by using a variety of
physicochemical remediation methods often involves excavation and land
filling, washing, replacement of soil with clean materials, or capping the soil
with an impermeable layer to reduce exposure to pollutants (Brown et al.,
2005).
There are many studies get different traditional methods to clean up
contaminated soils. Ho et al. (1995) use an injector to introduce hydrogen
peroxide below grade in an upward flow design for the remediation of soils.
Gopalan et al. (1993) used chemical treatment through design and synthesize
organic chelators for selective binding of actinide ions from soils and waste
streams. Tixier et al. (1992) have investigated the use of in-situ verification for
the remediation of pits and trenches used to dispose radioactive liquid wastes.
Luey et al. (1992) have demonstrated a large-scale in-situ verification process
on a site with heavy metal and radionuclide contamination that also contains
combustible timbers. Laboratory experiments have shown the efficiency of the
process for the removal of a wide variety of heavy metals (Lageman et al.,
1989; Acar and Alshawabkeh, 1993; Li et al., 1997, and Chung and Kang,
1999), radionuclide (Alshawabkeh, 1992), compounds (Schwartz et al., 1997)
and organic various media such as clays, sediments and saturated or unsaturated
soils.
Most importantly, some of these physicochemical engineering technologies
are proven to be economically unattractive particularly for large polluted sites,
and result in a considerable deterioration of the soil ecosystem (sometimes, they

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are even more damaging to the soil ecosystem than the pollutants themselves)
and do not permit a natural reshaping of the soil ecosystem (Lombi et al.,
2002). Taking into account that soil is one of our most important resources
(Pepper et al. , 2009), it is not surprising that there is a growing interest in the
development of environmentally friendly, and cost-effective methods for the
remediation of soils polluted with heavy metals.
1.3.4. Bioremediation
In contrast to traditional remediation approach, bioremediation a relatively
young; inexpensive and socially acceptable technology involves the use of
renewable resources like microbes and plants (phytoremediation) to tackle
heavy metal problems and subsequently to restore the lost fertility of soils
(Nies, 1999). It is an emerging technology and is viewed as the ecologically
responsible alternative to the environmentally destructive physicochemical
remediation methods (Meagher, 2005).
In general, the term bioremediation defines “a managed treatment process
that uses microorganisms to degrade and transform chemicals in contaminated
soil, aquifer material, sludge and residues” (Dasappa and Loehr, 1991). While
the phytoremediation is “the use of green plants to remove pollutants from the
environment or to render them harmless” (Cunningham and Berti, 1993).
Mixing the use of resistant plants and the application of microorganisms with
their beneficial effects to plants and to soil could represent a valid tool for soil
remediation (Wenzel et al., 2009). It should never be forgotten that the ultimate
goal of any soil remediation process must not be only to remove the
contaminants from the polluted soil, but, most importantly, to restore the
continued capacity of the soil to perform or function according to its potential
(i.e. to recover soil quality) (Epelde et al., 2009a). Among the various
bioremediation options, many scientists spread over different countries have
used live or dead culture of bacteria (Gutnick and Bach, 2000), fungi (Dhankhar

$*-!+))(!
9
and Hooda, 2011) yeast (Ruta et al., 2010) and algae (Poole and Gadd, 1989) to
biosorb heavy metals. Suhaimi et al. (2013) studied the biosorption of Pb (II)
ions from aqueous solution by treating corn (Zea mays) leaves biomass, and
founded that, the Z. mays is suitable and potential for removal of Pb from
aqueous solution. Oves et al., (2013) examined the metal biosorption ability of
B. thuringiensis strain OSM29, in the presence of the varying concentrations
(25 -150 mg/l) of heavy metals, such as Cd, Cr, Cu, Pb, and Ni. This strain
showed an obvious metal removing potential. Marais (2012) characterized
different bacterial species from platinum mine and determined their ability to
accumulate heavy metals inside the cells. Emili (2011) studied
phytoremediation of heavy metals which is enhanced by microorganism and the
results showed that this soil-plant-microorganism system was able to reduce the
total content of heavy metals by the average of 17% in the polluted soil. De
Jaysankar et al. (2008) studied several marine bacteria highly resistant to Hg
and tested them to evaluate their potential to detoxify Cd and Pb. Dursun (2006)
studied the biosorption capacity of Aspergillus niger and results showed the
maximum biosorption capacities were 28.7 and 32.6 mg /g at 250 mg dm-3
initial Cu (II) and Pb (II) concentration at 35°C respectively. Chojnacka et al.
(2005) reported the biosorption performance of Cr+3
, Cu+2
, and Cd+2
ions by
blue-green algae Spirulina sp. Al-Musawi (2010) studied ability of some some
species of Actinomycetes to biosorption of some heavy metals from from water
of eastern part of Al – Hammar marsh. Ahmed (2007) studied ability of eight
species of fungi to remove Hg from the soil. Angam and Israa (2006) use
Pseudomonas aeruginosa to Detoxification of Lead and Mercury Elements in
laboratory.
1.3.5. Metals contamination and microorganisms
Soil is an extremely complex environment that contains more microbial
genera or species than any other habitat (Kang and Mills, 2006) and the number
of species present in soil depends on the conditions available for their survival

$*-!+))(!
10
and growth (Stotzky et al., 1997). There are major interactions between the
different organisms using it as a habitat and any environmental disturbances or
changes that might prevail (Marais, 2012). The first organisms in the soil
environment to be influenced and to adapt to these changes are usually
microorganisms (Silver and Phung, 2005).
Microorganisms are usually highly adaptable to environmental changes.
Conditions such as temperature fluctuations, pH, salinity, carbon, energy
sources, and available water may affect species composition and could either
stimulate or inhibit microbial growth (Stotzky et al., 1997).
Environmental changes make it necessary for organisms to adapt and
develop tolerance to the various stresses in order to survive.
Microorganisms have developed resistance mechanisms which are either
chromosomal or plasmid driven (Malik, 2004). Microorganism's tolerance to
metals is accomplished by two kinds of actions. The first possibility is through
intrinsic properties that are related to the cell membrane structure such as
extra-cellular polypeptides that bind to metals and cause precipitation
(Vullo et al., 2008). Another way for microorganisms to adapt is to develop
specific mechanisms to deal with metal accumulation in cells, such as efflux
pumps and intracellular sequestration (Marais, 2012). Some strains can cause
the enzymatic transformation of metals and metalloids through oxidation. Metal
precipitation is used to immobilize metals to a lower redox state, producing a
less bioactive state which is often employed in wastewater treatment processes
(Valls and de Lorenzo, 2002). Metal biosorption where metals are bound to
cellular parts which is also a very useful process, especially where metals are
high in concentration, such as in effluents from industrial areas (Yilmaz, 2003).
1.3.6. Bioremediation of Metals by Microbial Processes
Bioremediation of metals is achieved through biotransformation. There are at
least three major microbial processes that influence the bioremediation of
metals:

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11
1- Biosorption and bioaccumulation.
Biologically catalyzed immobilization.2-
3- Biologically catalyzed solubilisation.
Biosorption and Bioaccumulation: Biosorption is the attract of the
positively charged metal ions to the negatively charged cell membranes and
polysaccharides secreted in most of the bacteria on the outer surfaces through
slime and capsule formation. While bioaccumulation is the retention and
concentration of a substance by an organism. The metals are transported from
the outside of the microbial cell through the cell membrane and into the cell
cytoplasm. The metal is sequestered and becomes immobile inside the cell (Losi
et al., 1994).
Many studies pick up biosorption and bioaccumulation ability of different
bacterial species. Gawali et al. (2014) studied the biosorption of three metals
(Cd, Zn and Cu) by an extracellular polymer substance (EPS) produced by
Pseudomonas sp, the results showed that the EPS is efficient to adsorb Cd, Zn
and Cu from the system. Ahmed and Malik (2014) studied the Zn accumulation
ability of three isolated from Pseudomonas sp (SN7, SN28, and SN30). Sinha
and Paul (2014) studied the accumulation of three metals (Pb, Cr and Cd) by
Aerococcus sp Chompoothawat et al. (2010) studied removal of Cd+2
from
aqueous solution by exopolysaccharid producing bacterium Ralstonia sp, and
the result proclaims that the Cd removal efficiency and Cd adsorption capacity
by EPS produce by this bacteria were 0.69 mg/g and 1.91%respectively.
Zolghrnein et al. (2010) studied the accumulation of heavy metals by
P.aeruginosa strain MCCB isolated from the Arabian Gulf. Salehizadeh and
Shojaosadati (2003) studied the removal of Pb, Cu and Zn using B. firmus and
they found that the removal efficiencies 98.3 %, 74.9 % and 61.8%
respectively. Stationary cells of B. thuringiensis exhibited high potential for Cd
binding (El-Helow et al., 2000).

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12
1.3.7. Examples of bacteria used in bioremediation of heavy metals
Bacteria are the most available and variable of microorganism and form a
signiﬁcant fraction of the entire living terrestrial biomass of ~1018
g (Mann,
1990). In early 1980 they found some microorganisms have the ability to
accumulate metallic elements with high capacity (Vijayaraghavan and Yun,
2008). Some marine microorganisms enriched Pb and Cd by factors of 1.7×105
and 1.0×105
respectively, relative to the aqueous solute concentration of these
elements in the environment (Mann, 1990). Bacteria were used as bioremediates
because of their small size, their ubiquity, their ability to grow under controlled
conditions, and their flexibility for a wide range of environmental condition
(Urrutia, 1997).
1.3.7.1. Deinococcus radiodurans
D. radiodurans is one of the most radiation resistant organisms known. It
can live in cold, dehydration, vacuum and acid, so it is known as a
polyextremophile and has been listed as the world's toughest bacterium in the
Guiness Book of World World Records (http. www. en. wikipedia. org
/wiki/Deinococcus radiodurans). D. radiodurans is a rather large, spherical
bacterium, with a diameter of 1.5 to 3.5 μm. Four cells normally stick together,
forming a tetrad. The bacteria are easily cultured and do not appear to cause
disease (Makarova et al., 2001). Colonies are smooth, convex, and orange to red
in color. The cells are stained gram positive, although its cell envelope is
unusual and is resemble of the cell walls of gram negative bacteria (Battista,
1997). This is due to its multilayered structure and lipid composition. At least
six layers have been identified by electron microscope (Rothfuss et al., 2006). It
does not form endospores and is non mobile. It is an obligate aerobic
chemoorganoheterotroph, (i.e., It uses oxygen to derive energy from organic
compounds in its environment)It is often found in habitats rich in organic
materials, such as soil, faeces, meat, or sewage, but has also been isolated from

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13
dried foods, room dust, medical instruments and textiles (Battista, 1997). As it
is considered as the major model for radiation resistance, the genome of D.
radiodurans (ATCC BAA-816) has been sequenced, and its genome consists of
two circular chromosomes, one is 2.65 million base pairs long and the other is
412,000 base pairs long, as well as a megaplasmid of 177,000 base pairs and a
plasmid of 46,000 base pairs. It has about 3,195 genes. In its stationary phase,
each bacterial cell contains four copies of this genome. When rapidly
multiplying, each bacterium contains 8-10 copies of the genome. D.
radiodurans can survive gamma irradiation doses that introduce hundreds of
double - strand breaks in its genome. The kinetics of DNA double - strand break
repair is very rapid as an intact genome complement is reconstructed from a
myriad of fragments in a few hours (Blasius et al., 2008).
1.3.7. 1.1. Application of D. radiodurans in bioremediation
The nuclear wastes typically implicate inorganic and organic contaminants
that contain radionuclides, heavy metals, acids/bases and solvents. The nuclear
wastes are predominantly contaminated with radionuclide's such as uranium,
plutonium, cesium, organo-pollutants (e.g. toluene, benzene, ethylbenzene,
xylene etc.), and heavy metals (Pb, Hg, Cr, As and Cd)
(http://www.lbl.gov/NABIR; Daly, 2000). The high radiation levels, to gather
with the chemical hazards, result in extreme damage to ecosystem and living
organisms.
The development of bioremediation strategies using Deinococcus sp.,the
members which are among the most radiation resistant organisms known, is
therefore vital for the cleanup of radioactive waste sites. Additional advantages
of deinococci in this field are that they are vegetative, easily cultured, and non-
pathogenic.
There are a lot of studies about the bioremediation ability of D. radiodurans
and its genetic engineering, for cleaning up heavy metals in nuclear west

$*-!+))(!
14
contaminated sites. Ginn and Fein (2008) studied proton, Pb, and Cd adsorption
onto the D. radiodurans, Thermus thermophilus, Acidiphlium. Feng et al.
(2008) studied the biosorption ability of D. radiodurans for radiocesium. They
noted that the maximum biosorption capacity of radiocesium by D. radiodurans
in equilibrium state was about 2, 100 kBqP/ kg (fresh weight basis). Appukuttan
et al. (2006) constructed D. radiodurans strain harboring phoN: a gene
encoding nonspeciﬁc acid phosphates obtained from a local isolate of
Salmonella enterica serovar typhi. The engineered strain retained uranium
bioprecipitation ability even after exposure to 6 kGy of Co60 gamma rays.
Brim et al. (2006) have reported that the engineered D. radiodurans, cloned
with tod and xyl genes of P. putida, is capable of complete degradation of
organic contaminants. Brim et al. (2000) have generated D. radiodurans strains
expressing the cloned Hg+2
resistance gene (merA) from E. coli BL308, MerA
encodes mercuric ion reductase, which reduces highly toxic, thiol-reactive
mercuric ion, Hg+2
to less toxic and inert elemental and volatile Hgo
, so that the
strains were shown to grow in the presence of both radiation and ionic mercury
at concentrations well above those found in radioactive waste sites, and to
effectively reduce Hg+2
to less toxic volatile elemental mercury. Fredrickson et
al. (2000) studied the reduction of Fe (III), Cr (VI), U (VI), and Tc (VII) by D.
radiodurans R1. The results proved that D. radiodurans can reduce Fe (III)
coupled to the oxidation of lactate or other organic compounds. Lange et al.
(1998) constructed strains of D. radiodurans that expressed toluene
dioxygenase activity. The resulting D. radiodurans constructed could oxidize
toluene, chlorobenzene, 3,4-dichloro-1 butene, and indole. The engineered
strain also grows and synthesizes toluene dioxygenase while being exposed to
ionizing radiation at a dose of 60 Gy h21.

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15
1.3.7.2. Shewanella oneidensis
S. oneindensis (formerly Shewanella putrefaciens) is a gram negative
bacterium, a facultative bacterium, capable of surviving and proliferating in
both aerobic and anaerobic conditions, straight or curved rod, 0.5–0.6 2–3
μm. Endospores and microcysts are not formed motile by a single, unsheathed,
polar ﬂagellum. Colonies are often pale tan to pink- orange, due to cytochrome
accumulation. No diffusible pigments are formed. NaCl is not required for
growth. Grows between 4 and 40°C; optimum temperature 30°C, oxidase and
catalase positive, chemoheterotrophic. Oxygen is used as the electron acceptor
during aerobic growth (Holt et al., 2005).
1.3.7.2.1. Application of S. oneidensis in bioremediation
Facultative bacterium S. oneidensis is able to use many organic carbon
sources as electron donors (lactate, pyruvate, propionate, acetate, fumarate, and
serine) and can reduce a variety of soluble or solid compounds, including iron
III, manganese I, nitrate, nitrite, thiosulfate, trimethyl-amine N-oxide, fumarate,
uranium, and Cr (VI) (Scott and Nealson, 1994; Venkateswaran et al., 1999 and
Tiedje, 2002). Mechanisms responsible for metal oxide reduction are not fully
understood, but it is clear that a number of genes are involved. These include
the mtrA, mtrB, and mtrC genes (Beliaev et al., 2005).
And because of its metabolic versatility and its ability to reduce metals to
less mobile, there have been extensive studies of this bacterium, primarily
focused on its versatile respiration and its potential to engage in co metabolic
bioremediation of toxic metals oxide forms (Tiedje, 2002; Viamajala et al.,
2002, 2004; Middleton et al., 2003 and Tang et al., 2006). This bacterium has
been considered for use in bioremediation of subsurface sites contaminated with
metals and, as such, has been studied extensively over the last decade (Myers
and Nealson, 1988; Viamajala et al., 2002, 2004; Abboud et al., 2005).

$*-!+))(!
16
The effects of humic acid (HA) on azo dye decolorization by S. oneidensis
MR-1 were studied by Liu et al. (2011). They found that, 8% of the Pu (IV) was
reduced to Pu (III) after 24 h. Renshaw et al. (2009) studied the amount of Pu
reduced is less (3%) and is cell-bound.
Bretschger et al. (2007) studied S. oneidensis (wild and some cytochrome
mutant one) for studying current production and metal oxide reduction. Cruz-
Garcia et al. (2007) studied respiratory nitrate ammoniﬁcation by S. oneidensis
MR-1. The result showed anaerobic cultures of S. oneidensis MR-1 grown with
nitrate as the sole electron acceptor exhibited a sequential reduction of nitrate to
nitrite and then to ammonium. Hong et al. (2007) reported that azo dye
decolorization by S. oneidensis S12 was accelerated by “humic substances”
acting as redox mediator. Boukhalfa et al. (2007) also investigated the reduction
of Pu (IV) by S. oneidensis with lactate as the electron donor. Middleton et al.
(2003) studied the reduction of Cr (VI) by S. oneidensis under aerobic and
denitrifying conditions and in the absence of an additional electron acceptor,
and also has been described by Fein et al. (2002).
1.3.7. 3. Bacillus thuringiensis
B. thuringiensis is a ubiquitous, gram-positive and spore-forming
bacterium, similar to other Bacillus species in morphology and shape (Stahly et
al., 1991). The organism is a facultative anaerobe. The cell has rod shape, and
the width of the rod is 3-5 μm in size when grown in standard liquid media. The
spore formation of the organism varies from terminal to subterminal in
sporangia. Colony morphology can help to distinguish B. thuringiensis
colonies from other Bacillus species. The organism forms white, rough
colonies, which spread out and can expand over the plate very quickly. The
major distinguishing feature of B. thuringiensis from closely related Bacillus
species (e.g. B. cereus, B. anthracis ) is the presence of a parasporal crystal

$*-!+))(!
17
body that is near to the spore, outside the exosporangium during the endospore
formation, (Andrews et al., 1985; Andrews et al., 1987 and Bulla et al., 1985).
B. thuringiensis was first isolated by Sotto Ishiwata in 1901 from a diseased
silkworm larvae (Bombyx mori) and named the isolate as Bacillus sotto. It was
not characterized until a decade later. E. Berliner isolated a similar Bacillus
from a diseased Mediterranean flour moth larvae (Anagasta kuehniella), and
named his isolate as B. thuringiensis (Cannon, 1995).
1.3.7. 3.1. Application of B. thuringiensis in bioremediation
B. thuringiensis has multiple heavy metal resistance phenotypes, and
considerable cell surface affinity for metal cations and the ability to express a
variety of extracellular digestive enzymes (Amer, 1996). These advantageous
characteristics provide promising prospects for future environmental protection
studies. It seems likely that, this bacterium can be tailored for efficient growth
in metal-polluted environment supplemented with inexpensive nutrients, which
might include by-products and wastes, resulting in bioremediation with
simultaneous secretion of commercial extracellular enzymes (El-Helow et al.,
2000).
For the above reason, there are many studies to use this bacterium in heavy
metal bioremediation. Thamer et al. (2013) studied the ability of B.
thuringiensis in biodegradable of light crude oil. These bacteria exhibit the
ability to dismantle crude oil through clear emulsion layers of crude oil. Oves et
al. (2013) use strain OSM29 of B. thuringiensis to study the biosorption ability
of these bacteria, The biosorption capacity of the strain OSM29 for the metallic
ions was highest for Ni (94%) which was followed by Cu (91.8%), while the
lowest sorption by bacterial biomass was recorded for Cd (87%) at 25mgl-1
initial metal ion concentration. Marandi (2011) was investigated the biosorption
of Cu and Mn ions from Sarcheshme copper mine wastewater by a locally
available bacterium B. thuringiensis, was investigated in batch mode.

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18
Rathnayake et al. (2010) isolated B. thuringiensis as bacteria have the ability to
tolerate heavy metals in pristine soil. Ozturk (2007) studied the biosorption of
the toxic metal (nickel) from aqueous solutions on dried vegetative cell and
spore–crystal mixture of B. thuringiensis. In other work B. thuringiensis 4G1
was used to decolonization ethylene blue. A verification experiment performed
under optimal conditions yielded 98.23% of the predicted decolonization %
(100%) with an increase by factor 1.3 compared with the result obtained under
basal conditions (El-Sersy and El-Sharouny, 2007). Sahin and Ozturk (2005)
studied the biosorption of Cr (VI) ions from aqueous solution by drying
vegetative cell and spore–crystal mixture of the bacterium B. thuringiensis.
From the results they, recorded that, at the optimal conditions, metal ion uptake
has raised with increasing initial metal ion concentration. Cr (VI) ions uptake of
B. thuringiensis spore–crystal mixture at 250 mg l-1
was 24.1%, whereas it's
vegetative cell metal uptake was 18.0%.

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19
2. Materials and Methods
2.1. Materials
2.1.1. Chemicals
Table 1: All chemicals used in the study
OriginSupplierChemicals
SpainScharlouAcetic acid
USASigmaAmmonium nitrate
UKBDHBromo phenol blue
IndiaHi mediaCacodylate
IndiaHi mediaCadmium chloride
IndiaHi mediaCalcium chloride
IndiaHi mediaChloroform
UKBDHChromic acid
UKBDHCrystal violate
UKOxoidCasein hydrolysis
UKOxoidDextrose
UKBDHDimethylaminobenzaldeyde
UKBDHDimethyl-P-
Phenylenediamine
UKBDHDiphenyl amine
SpainScharlouDisodium hydrogenortho
phosphate
IndiaHi mediaEDTA
SpainScharlouEthanol

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20
IndiaHi mediaFerrous ammonium sulfate
UKOxoidGelatin
IndiaHi mediaGlucose
IndiaHi mediaGulataraldehyde
SpainScharlouGlycerol
IndiaHi mediaHydrochloric acid
SpainScharlouHydrogen peroxide
UKBDHIodine
IndiaHi mediaIsoamyl alcohol
USASigmaIsopropanol
IndiaHi mediaLead nitrate
IndiaHi mediaMagnesium chloride
IndiaHi mediaMannitol
UKBDHMethyl red
UKBDHN, N-
dimethylnaphtholamine
IndiaHi mediaOsmium tetroxide
IndiaHi mediaOxidase reagent
GermanyRoche Diagnostic
GmbH
PCR purification kit
IndiaHi mediaPerchloric acid
UKBDHPhenol red
SpainScharlouPolyethylene glycol
UKBDHPotassium chloride
IndiaHi mediaPotassium dichromate

$*-!+--0)
21
USASigmaPotassium di hydrogen
phosphate
IndiaHi mediaPotassium hydroxide
IndiaHi mediaPotassium nitrate
UKOxoidProteose peptone
UKBDHSafranin
IndiaHi mediaSodium chloride
IndiaHi mediaSodium citrate
IndiaHi mediaSodium hydroxide
UKOxoidStarch
IndiaHi mediaSucrose
UKBDHSulfanilic acid
UKBDHSulforic acid
TaiwanYeastren BiotechTaq DNA polymerase
USAPromegaThe Wizard®Genomic
DNA purification kit
USASigmaTrihydrogen phosphate
IndiaHi mediaUranayal acetate
2.1.2. Equipments
Table 2: All Equipments used in the study
OriginSupplierEquipments
JapanHirayamaAutoclave
UKBaird and TatlockCentrifuge
GermanySartoriusCooling Shaker
incubator

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22
GermanyLeicaDigital Microscope
GermanySartoriusDigital balance
JapanHitachiField emission electron
microscope
GermanySartoriusFiltration unit
UKPhoenix-986-BiotechFlame atomic
spectroscopy
JapanShimadzuFourier transform
infrared
GermanyChristFreeze drying
GermanyHeidolphHot plate and Magnetic
stirrer
GermanyBinderIncubator
UKBassairLaming air flow
GermanyZeissMicroscope
GermanyBinderOven
USABio- RadPCR machine
GermanyLovibondpH meter
JapanHitachiSpectrophotometer UV-
Vis
JapanHitachiTransmission electron
microscope
GermanyHeidolphUni vortex
GermanyGFLWater bath
JapanShimadzuX-ray powder
Diffractrometer

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23
2.1.3. Media and media components
Table 3: Media and media components
OriginSupplierMedia
IndiaHi mediaAgar
UKOxoidBeef extract
IndiaHi mediaFerrous sulfate hydrate
GermanyMerckLuria-Bertani broth
GermanyMerckLuria-Bertani agar
IndiaHi mediaLuria-Bertani agar
UKOxoidMethyl Red and Voges
Proskauer
UKOxoidNitrate broth
IndiaHi mediaNutrient broth
IndiaHi mediaNutrient agar
UKOxoidOxidation fermentation
Medium
UKOxoidPeptic digested animal
tissue
UKOxoidPeptone
UKOxoidProteose peptone
UKOxoidSimmons citrate agar
UKOxoidTriple sugar iron agar
(TSI)
UKOxoidTryptone
UKOxoidTryptone water broth
UKOxoidYeast extract

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24
2.1.4. Isolation media
All media have been autoclaved at 121 °C, under 1.5 bars for 15 min.
2.1.4.1. BYS MEDIUM (Shivajie et al., 1988)
It consists of 5 g peptone, 1g yeast extract, 5 ml soil extract, and 1.5 g agar
per 1000 ml of distilled water, pH 8. Used as a selective medium for isolation D.
radiodurans.
2.1.4. 2. M B MEDIUM (Medium B) according to (Ivanova et al., 2003).
It consists of 0.2g peptone, 0.2g Casein hydrolysate, 0.2g yeast extract, 0.1g
glucose, 0.02g KH2PO4, 0.005g MgSO4.7H2O, and 1.5g agar per 100 ml
distilled water, pH 7.5. Used as a selective medium for isolation S. oneidensis.
2.1. 5. Biochemical Tests Media
2.1.5.1. Nitrate reduction medium (Harley and Prescott 1996)
It consists of 3gm beef extract, 5gm peptone, 1gm potassium nitrate, per1000
ml distilled water, pH 7.0. The medium was used for determination of nitrate
reduction.
2.1.5.2. Buffered peptone – glucose broth medium (commercially available
)2013,Reiner()VP broth-as MR
It consists of 7gm peptone, 5gm K2HPO4, and 5gm dextrose. Per 1000 ml
distilled water, pH 6.9. The medium was used for both methyl red and Voges-
Proskauer tests.
2.1.5.3. Tryptone water broth medium
It was purchased from Lab M. Wash Lane, Bury, BL 9, AV. England and
used for indole test.
2.1.5.4. Nutrient gelatin (Leboffe and Pierce, 2010)
This medium used for gelatin liquefaction test, It consists of 5 gm peptone,
3gm beef extract, 12gm gelatin, per 1000 ml distilled water, pH 6.8. Used for
gelatin liquefaction test.

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25
2.1.5.6. Starch agar medium (Collins et al., 1995)
It consists of 3gm beef extract, 10gm soluble starch, and 12gm agar, per
1000ml distilled water. The medium was used for starch hydrolysis.
2.1.5.7. Phenol red carbohydrate broth (Bartelt, 2000)
It consists of proteose peptone 10gm, sodium chloride 5gm, 1gm beef
extract, 0.018 gm phenol red, and 10 gm carbohydrate, per 1000ml distilled
water; (pH 7.4 ± 0.2). Used with carbohydrates for the differentiation of
microorganisms on the basis of carbohydrate fermentation reactions.
2.1.6. Reagents
2.1.6.1. Nitrate Reduction Reagent (Harley and Prescott, 1996)
It consists of two reagents: A consists of 4 gm sulfanilic acid dissolved in
500 ml acetic acid. Reagent B consists of 2.5 gm alpha- naphthylamine.
Dissolved in 500 ml acetic acid (0.5N). This reagent was used to detect the
presence of nitrite produced by the reduction of nitrate in nitrate broth.
2.1.6.2. Oxidase Reagent
It was purchased from Hi media (India). The reagent used to perform the
cytochrome c oxidase test.
2.1.6.3. Catalase Reagent (Harley and Prescott, 1996).
A concentration of 3% of hydrogen peroxide was used to detect the ability of
bacteria to produce catalase.
)2013,Reiner(s reagent’Kovac4.6..12.
It was purchased from Hi media -India. It is used for the diagnostically indole
test

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26
2.1.6.5. Voges – Proskauer reagents
It consists of:
Reagent A, alpha-naphthol 50gm
Absolute ethanol 1000 ml
Reagent B, potassium hydroxide 400gm
Deionized water 1000 ml
Voges Proskauer reagents are recommended for use in determining the
presence of acetone as intermediate in 2,3 butanidaol fermentation pathway
)2013,Reiner(reagentMethyl red solution6..6.12.
It is prepared by dissolving 0.1g of methyl red in 300ml of ethanol (95%).
Add 200ml of deionized water to make 500ml of 0.05% (w/v) solution. Is an
indicator dye that turns red in acidic solutions.
2.1.6.7. Potassium dichromate 1N reagent (Page et al., 1982)
It is prepared by dissolving 40.04 gm of K2Cr2O7 in distilled water and
making up to 1 liter. Use in determining the TOC.
2.1.6. 8. Ferrous-ammonium-sulfate, 0.4N reagent (Page et al., 1982)
Prepared by dissolving 159.6 gm of Fe (NH4)2(SO4)2.6H2O in distilled
water containing 40 ml concentrated H2SO4 and making up to 1liter. Determine
normality periodically by titration against the potassium dichromate solution.
Use in determining the TOC.
2.1.6. 9. Diphenyl indicator solution reagent (Page et al., 1982)
Dissolving 0.2 g of diphenyl amine in 100ml concentrated H2SO4 storing in
the glass dropping bottle. Use in determining the TOC.

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27
2.2. Methods
2.2.1. The Study Area:
The study area included three different districts in Basra city south of Iraq.
Samples were collected during January – 2013(Fig 1).
1. Fao; located about 90 Km south of Basra city.
2. Um Qasr; located about 60 Km south west of Basra city.
3. Al-Zubair; located about 20 Km west of Basra city.

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28
Fig1. A map shows the sites of sampling

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29
2.2.2. Soil sampling and handling
A total of 9 composite soil samples was collected in 0-20 cm depth from
three different selected areas.
The soil samples were labeled and stored in plastic bags and taken to the
laboratory. In the laboratory, each sample was separately dried in air and milled
using porcelain pestle and mortar, then sieved with 2 mm sieve. The fine soil
fractions are collected in separate bags, and store in a dry place to use in further
analysis.
2.2.3. Soil analyses
The soil samples were analyzed for various physical and chemical
characteristics such as soil texture, pH, EC, total organic carbon, and total
concentration of heavy metals.
2.2.3.1. Soil texture
It was done by using the pipette method according to Black (1965).
2.2.3.2. pH
About 50 gm of each soil sample was taken in a glass beaker and 100 ml of
distilled water (1:2) was added. The contents were mixed with shaker and
allowed to stand for one hour. The soil pH was measured using calibrated
Lovibond pH 200 meter (Page et al., 1982).
2.2.3.3. Electrical conductivity (EC)
Three hundred grams of each soil sample were taken in a glass beaker and
soil saturated paste was prepared. Soil extract was obtained from the saturated
soil paste using a vacuum pump. EC was recorded using calibrated Lovibond
con200 meter (Page et al., 1982).
2.2.3.4 .Total organic carbon (Organic C)
Two grams of soil were taken in a 500 ml Erlenmeyer flask. 10 ml of 1 N
K2Cr2O7 was added and the flask was agitated to mix the contents. Twenty ml

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30
of conc. H2SO4 was added to the soil suspension. The flask was agitated again
for 1 min, and allowed to stand for 30 min. After that, 200 ml of distilled water,
10 ml of H3PO4 and 1 ml of diphenylamine indicator were added and the
contents were titrated against 0.5 N FeSO4.7H2O until the color changed from
blue to red (Page et al., 1982).
2.2.3.5. Total concentration of heavy metals in soils (Emili, 2011)
In the present study, the total contents of Pb and Cd were determined in all
soil samples. The soil (1g from soil dry in oven for 5 days) was mixed with 5 ml
of nitric acid (HNO3) and 2 ml of perchloric acid (HClO4); and then put in a
Block digester with the following cycle of time and temperature: 2 hours at 90
°C, 2 hours at 140 °C and 1 hour at 190 °C. Then, the extracts were made to
volume (50 ml) with deionized water, filtered and analyzed by flame atomic
absorption spectrophotometer.
2.2.3.6. Heavy metal bioavailability
CHCl3labile Pb and Cd
All soil samples are subjected to a preliminary incubation at 60% water
holding capacity (WHC) for 7 days (Brookes et al., 1995). Twelve sub
samples of 10 gm moist soil were taken from each soil, and extracted with 25
ml of 1 M NH4NO3 (1:2.5 w: v), shaked for 60 min at 200 rpm and filtered
through paper (Whatman No. 42). Twelve replicates were fumigated for 24 h
with ethanol -free chloroform (CHCl3) in vacuum sealed desiccators at room
temperature. Following fumigant removal, these samples were extracted as
described in the non-fumigated replicates. After filtration, the soil extracts were
acidified with 0.5 ml 65 % HNO3 and stored at 4°C. Then, in all extracts, Pb
and Cd were measured by flame atomic absorption. CHCl3 labile Pb and Cd
were calculated as the difference between Pb or Cd extracted from fumigated
soil and those extracted from non fumigated soil (Khan and Joergensen 2009).

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2.2.4. Isolation of bacteria
Each one gram of the soil sample was suspended in 99 ml of sterile distilled
water and shaken vigorously for 2 min serially decimal diluted supernatant in
sterile distilled water of 10-1
to 10-5
was plated on isolation medium for each
type of study bacteria. Plates were incubated at 30°C for 24h.

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2.2.5. Identification of bacteria
Flowchart (1) describes the procedure used for isolation and identification of
bacteria obtained in this study.
Isolation source
Soil
Isolation of Bacteria
Purification of bacteria
Identification of bacteria
Molecular
Biochemical testes
Microscopic
Examination
As in section 2.2.5.1.
Oxidase
Morphology
Motality
Gram stain
Citrate utilization
H2S production
Voges-Prosker
Hydrolysis of starch
Carbohydrate
fermentation
Carbohydrate
fermentation
Nitrate reduction
Deinococcus radiodurans
Shewanella oneidensis
Bacillus thuringiensis

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2.2.5.1. Molecular identification
This study was done in Cell and Molecular Biology Lab, College of
Biotechnology, University Putra Malaysia.
2.2.5.1.1. Total genomic DNA extraction
Total genomic DNA of all bacterial samples was extracted using
Wizard® Genomic DNA Purification Kit (Promega, USA) following the
manufacturer’s instruction. Briefly, overnight-grown bacterial culture was
harvested at 13,000 rpm for 1 minute. The harvested pellet was suspended in
480μl 50mM EDTA, then added with 120μl lytic enzyme(s) before being
incubated at 37°C for 60 minutes. The mixtures were then centrifuged for 2
minutes at 13,000 rpm and the supernatant was removed. To the pellet, 600μl
Nuclei Lysis Solution was added and mixed gently by pipetting. The mixture
was then incubated for 5 minutes at 80°C and let cool to room temperature.
Three microliters of RNase Solution was added to the mixture and incubate at
37°C for 15–60 minutes, then cool to room temperature. To the mixture, 200μl
of Protein Precipitation Solution was added, vortexes and incubated on ice for 5
min, followed by centrifugation at 13,000rpm for 3 minutes. The supernatant
was transferred to a clean tube containing 600μl of room temperature
isopropanol and mixed properly. The mixture was centrifuged for 2 minutes at
13,000 rpm, and the supernatant decanted. Six hundred microliters of room
temperature 70% ethanol was added, mixed and centrifuged for 2 minutes at
13,000rpm. The ethanol was aspirated and the pellet was air-dried for 10–15
minutes. The DNA pellet was dehydrated in 100μl of Rehydration Solution for
1 hour at 65°C or overnight at 4°C.

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2.2.5.1.2. 16 S rRNA
Bacterial samples were identified by sequencing of the 16S rRNA gene. To
determine the identity of the bacterial samples, the amplified 16S rRNA gene
PCR products obtained from total genomic DNA using primer Set: 27F (5′-
AGAGTTTGATCCTGGCTCAG-3′),1492R(5′-GGTTACCTTGTTACGACTT-
3′), (Lane et al., 1985) were sequenced commercially. PCR was carried out in a
100 L reaction mixture containing 10 L of 10X PCR reaction buffer, 10 L
of 10X 27F/1492R 2.5 M each, 2 L of 10 mM PCR–grade of
deoxynucleoside triphosphate (dNTP), 30 ng/L of DNA template, 62 L of
sterile Milli–Q water and 1 L (5 U/L) of Taq DNA polymerase (Yeastern
Biotech Co. Ltd., Taiwan). The PCR was carried out in a Bio–Rad My Cycler
thermal cycler (Bio–Rad, USA) with an initial denaturation step at 95 °
C for 5
min, 35 cycles of 95 °
C for 30 s, 52 °
C for 30 s, 72 °
C for 1 min, followed by a
final extension step of 72 °
C for 10 min.
2.2.5.1.3. PCR products purification
PCR products were purified using high pure PCR product purification kit
(Roche Diagnostic GmbH, Germany) by following the manufacturer’s
instruction. Briefly, 5 volumes of Binding buffer to 1 volume of PCR product
was mixed well and transferred into high pure filter tube. The DNA was bound
to the filter by centrifugation at 13,400 rpm for 1 min. the flow-through was
discarded. An additional 500 μl of wash buffer was added and centrifuged at
13,400 rpm for 1 min. The flow-through was discarded. Additional
centrifugation at 13,400 rpm for 1 min was performed after 200 μl of wash
buffer was added. Finally, DNA was eluted into a clean 1.5-ml microcentrifuge
tube by adding 50 μl of Elution buffer (10 mM Tris-HCl) and centrifuged at
13,400 rpm for 1 min.

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2.2.5.1.4. Bioinformatics analysis of bacteria samples
DNA sequences obtained were compared to sequences available online in
the GenBank database (http://www.ncbi.nlm.nih.gov). Homology search was
performed by using bioinformatics tools available online, BLASTn
(http:/www.ncbi.nlm.nih.gov/BLAST) (Altschul et al., 1997).
2.2.6. Preparation of heavy metals concentrations
Stock solutions of the metal salts were prepared by dissolving the exact
quantities of Pb (NO3)2 and Cd (NO3)2.H2O in sterile deionized distilled water.
The working concentration of Cd (II) and Pb (II) solution was prepared from
suitable serial dilution of the stock solution according to (Etorki et al., 2013).
2.2.7. Determination of Minimum Inhibitory Concentrations (MIC) of
heavy metals
The disk diffusion methods have been used for determining the MIC of the
metals for each isolate (Wistreich and Lechtman, 1980). The concentrations of
Pb and Cd were between 40 to 2500 mg/l. Filter paper disks were saturated with
heavy metals for 30 min, and then added to nutrient agar plates which had been
cultured with bacteria. Plates were incubated at 30ºC for 24 h.
2.2.8. Bioaccumulation of heavy metals by bacteria
Bacteria were grown in LB broth containing different concentrations of Pb
(5 , 10 , 25 , 50 mg/l) and Cd (10 , 20 , 50 , 100 mg/l) for (2 , 4 , 6 , 24 and 48 h)
and incubated at 30ºC in a shaker incubator at 150 rpm. Three replicates for
each concentration have been done as, one control. The bacterial cells were
harvested by centrifugation at 6000 rpm for 15 min and suspended in 1 ml of
distilled water, oven – dried and weighted. Metal concentrations were measured
by atomic absorption spectrophotometer. Control was represented by the same
microbial culture without heavy metals. Each metals concentration is measured
with two replicates (Sprocati et al., 2006).

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2.2.8.1. Digestion of bacterial biomass for atomic absorption measurement
(Jiang, 1994).
The bacterial cells were harvested by centrifugation at 6000 rpm for 15 min,
washed with distilled water, and then dried at 60°C for 1h. The dried biomass
were then digested as follow: 100 ml beaker containing the dried cells, 5 ml
concentrated nitric acid was added; the beaker was placed on a hot plate, stirred
continuously, and heated initially at a medium rate for 5 min. Then, the beaker
was heated on maximum setting until nitrogen oxide fumes were given off for a
short time and a white residue was left. The beaker was left to cool for about 2
min and digestion was repeated with an additional 2 ml of concentrated nitric
acid; this time it was heated until brown nitrogen oxide fumes almost ceased to
appear. The beaker was cooled again for about 2 min and then 2 ml of 1:1
hydrochloric acid (35- 37%) was added. The mixture was heated at a medium
rate for 3 min. After that it was cooled to room temperature and made up to 25
ml or bigger volume with distilled water. These samples were analyzed by
atomic absorption spectroscopy.
2.2.9. Biosorption of heavy metals by Bacteria
The equilibrium kinetics data of the biosorbent bacteria were obtained by
performing batch experiments. The experiments were carried out in 250 ml
flasks to which 100 ml of heavy metals solution and l ml of biomass
(exponential phase) was added. The mixture was stirred at 150 rpm at 30 °C; 1
ml of each sample which was collected and centrifuged at 6000 rpm for 10 min.
The remaining concentration of both Pb and Cd in residual solution was
measured by flame atomic absorption. The final reading for each solution was
taken at intervals time 2, 4, 6, 24, and 48h. Each experiment was carried out
with two replicates and control (Sethuraman and Kumar, 2011).

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2.2.10. Characterization study
2.2.10. 1. Transmission electron microscope TEM/ Field Emission
Scanning Electron Microscopy (FE-SEM)
By centrifuging samples in suspension for 10 min at 300 rpm, and
decanting the supernatant, fixing pellet with 4% gutaraldehyde for 4h at 4°C
and centrifuged again, decanted fixative and adding an appropriate quantity
animal serum to submerge sample, and allowed serum to clot. It was washed
three times with 0.1M Cacodylate buffer for 10 min and Posted fix in 1%
Osmium tetrroxide for 2 h at 4°C. Also, it is washed again three times with
0.1M Cacodylate buffer for 10 min. Dehydrating in series of acetone (35, 50,
75, 95, and 100%) for 10, 10,10, 10 and 15 min respectively.
Finally, we make infiltration of the specimen with acetone and resin
Acetone Resin Time
1 : 1 1h
1 : 3 2h
100% resin Overnight
100% resin 2h
Embedding: specimens were placed into beam capsule filled with resin
Polymerization: polymerize in oven at 60 °C for 24-48h.
Make ultracectioning, by choosing an area of interest, then cut for ultrathin
section, selected the silver section, picked up a section with a grid then drying
with filter paper. Finally the section stained with Uranyl acetate for 15 min, and
washed double distills water. Lead stained for 10 min, and washed double in
distilled water. This analysis was done in the Electron Microscope Laboratory,
Institute of Bioscience, University Putra Malaysia

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2.2.10.2. FTIR analysis
The Fourier transform infrared (FTIR) analysis was done with Perkin
Elmer spectrometer model 100 series (sample preparation UATR). This analysis
was done at Chemistry Department, University Putra Malaysia.
2.2.10.3. X-ray powder diffraction analysis (XRD)
The powder X-ray diffraction analysis was performed using a Shimadzu
diffractometer model XRD 6000. The diffractometer employed Cu-Kα radiation
to generate diffraction patterns from powder crystalline samples at ambient
temperature. The Cu-Kα radiation was generated by Philips glass diffraction, X-
ray tube broad focus 2.7KW type. The crystallite size D of the samples was
calculated by using the Debye–Scherrer's relationship. Where D is the
crystalline size, λ is the incident X-ray wavelength, β is the (FWHM) Full
Width at Half-Maximum, and θ is the diffraction angle, Deby -Scherrer
equation can be written as: D= K λ / B COS θ.
This analysis was done at Chemistry Department, University Putra Malaysia.
2.2.11. Statical analysis
Statistical analysis was carried out using one way Analysis of Variance
(ANOVA) was used to compare means and significantly different means were
separated using LSD; with post test if P0.05 and using SPSS Ver.10 software

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39
3. Results
3.1. Physical and chemical properties of soil
3.1.1. Soil texture
In the studied sites two types of soil textures are recognized on the basis of
percentage of sand, silt and clay for division of soils into textural classes. In
general, the sand fraction (ranging from 1 - 76 %), the clay fraction (ranging from
3 - 24 %), while silt fraction ranged (21-75%). According to these combinations
the studied soils take different texture (Table 4).
Table 4: Soil texture of studied samples.
Soil Texture
Soil particle distribution
District Clay%Silt%Sand%
Silt loam24751Fao
Loam sand32176Um-Qasr
Silt loam65737Al-Zubair
3.1.2. pH, Electrical conductivity (EC), and Total organic carbon (TOC)
The pH measurements of the soils ranged between 8.27-8.56 (Table 5). The
EC was found at lowest value in Al-Zubair soil (2.30 ms/cm) while the highest
values were in Fao and Um-Qasr soils (10.50 and 9.84ms/cm) respectively. The
level of TOC was in the following order FaoUm-QasrAl-Zubair (Table 6). The
variance of analysis of data shows significant (P0.05) differences in pH, EC, and
TOC for all studied districts, as it was clear from LSD value (0.09, 0.67, and
0.105) respectively.

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Table 5: pH, Electrical Conductivity (ms/cm) and Total organic carbon (%)
in sample soils.
Total organic
carbon (TOC) (%).
E.C.
)ms/cm(
pHDistricts
0.5010.508.35Fao
0.209.848.56Um-Qasr
0.102.308.27Al-Zubair
3.2. Total concentration of heavy metals
During the period of study (January 2013) the total concentrations of Pb and
Cd in soil samples were measured (Table 6). The high concentration of Pb and Cd
were in Um-Qasr soil (28.39 and 6.81μg/g) respectively, followed by Fao soil
(19.80 and 5.96 μg/g) respectively, while the lowest concentrations were 14.16
and 4.30 μg/g respectively in Al-Zubair soil. So, the presence order of these
metals in these soils takes the series Um-Qasr Fao Al-Zubair. The variance of
analysis of data shows significant (P0.05) differences in total concentration of
both metals (Pb and Cd) in related to studied districts, as it was clear from LSD
value (5.64 and 0.84) respectively.
Table 6: Total concentration of Pb and Cd (μg/g) in soil samples.
Total concentration (μg/g)
Districts CdPb
5.9619.80Fao
6.8128.39Um-Qasr
4.3014.16Al-Zubair

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3.3. Bioavailability of studied heavy metals
Table (7) shows the availability of Pb and Cd in studying soils. In general the
availability of Pb and Cd was low in all studied soils in comparison with the total
concentration of these metals. While the Cd recorded more availability than Pb
in all these soils.The highest Cd availabilities were 0.69 and 0.41μg/g for Um-
Qasr and Fao soils, respectively, while the lower availability was 0.24μg/g in
Al-Zubair soil. In case of Pb, the highest availabilities were 0.28 and 0.17μg/g for
Um-Qasr and Fao soils, respectively, while the lowest was 0.11 μg/g in Al-
Zubair soil. The variance of analysis of data shows significant (P0.05)
differences in bioavalibility for both metals (Pb and Cd) in related to studied
districts, as it was clear from LSD value (0.06 and 0.16) respectively.
Table 7: Bioavailability (μg/g) of Pb and Cd in soil samples.
Bioavailability (μg/g)
District CdPb
0.410.17Fao
0.690.28Um-Qasr
0.240.11Al-Zubair
3.4. Isolation and Identification of bacteria
3.4.1. Deinococcus radiodurans
3.4.1.1. Cultivation characteristics, microscopic examination and
biochemical tests.
The bacterium was identified depending on the biochemical tests (Table 8),
and colony morphology of growing isolates on TGY agar medium. Colony size
appears middle to big and has orange color, while the shape is round and
convex (Pic. 1). Under the light microscope, bacteria are gram positive, non
motile, and usually four cells stick together, forming a tetrad (Pic. 2).

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D. radiodurans is a rather large, spherical bacterium, with a diameter of 1.5
to 0.8 μm (Pic. 3) as appeared under field emission scanning electron
microscope (FE-SEM).
Picture 1: Colony morphology of D. radiodurans grows on
TGY medium.
Picture 2: Microscopic shape of D. radiodurans (gram stains) (1000X).
Bacteria appear gram positive cocci and four cell form
tetrad.

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Picture 3: D. radiodurans as it appears under FE-SEM with cell
Table 8: Biochemical characteristics of D. radiodurans isolated from
soil.
Characteristics observedTests
+Oxidase test
+Catalase test
+Indole formation
+Citrate utilization
+Gelatin liquefaction
-Nitrate reduction
Fermentation
D-glucose
Mannose
Lactose
+
+
-
+Arginine
+Hydrolysis of casein
+and - indicate positive and negative reactions,
respectively

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3.4.2. Shewanella oneidensis
3.4.2.1. Cultivation characteristics, microscopic examination, and
biochemical tests.
Based on colonies morphology of growing isolates on MB agar bacterial
colonies were circular, smooth, and convex, and are often pale tan. No
diffusible pigments are formed (Pic. 4). Under the light microscope, bacteria are
gram negative, motile, and straight or curve rode (Pic. 5). Picture (6) represents
the bacterium under FE-SEM. Bacteria appear rod with diameter 1.91-0.71 μm.
Table (9) shows the biochemical tests used in identification
Picture 4: Colony morphology of S. oneidensis
grows on MB medium.

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Picture 5: Microscopic shape of S. oneidensis (gram stains)
(1000x). Bacteria appear gram negative curve rode.
Picture 6: S. oneidensis as it appears under FE-SEM, with cell

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Table 9: Biochemical characteristics of S. oneidensis isolated from
soil.
3.4.3. B. thuringiensis
3.4.3.1. Cultivation characteristics, microscopic examination, and
biochemical tests.
This bacterium forms a white rough colony when grown in L.B agar (Pic 7).
Under the light microscope, it appears gram positive and has a rod shape with
endospore (Pic. 8). The cell measurements rang 3-5 μm under FE-SEM (Pic. 9).
The bacteria have been identified by using biochemical characteristics (Table 10).
+Oxidase test
+Catalase test
-Indole formation
+Nitrate reduction
+Production of H2S
+Gelatin liquefaction
Fermentation of
Sucrose
Fructose
D-glucose
+
+
+
respectively

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Picture 7: Colony morphology of B. thuringiensis grows
on L.B medium.
Picture 8: Microscopic shape of B. thuringiensis (gram stains)
(1000X). Bacteria appear gram positive bacilli with terminal
spore.

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Picture 9: B. thuringiensis as it appears under FE-SEM, with cell
Table 10: Biochemical characteristics of B. thuringiensis isolated from
soil.
-Oxidase test
+Catalase test
+Indol formation
-Nitrate reduction
+Voges Proskauer
+Citrate utilization
+Methyl red
Carbohydrate utilization
Sucrose
D- glucose
Mannitol
+
+
-
-Hydrolysis of
+Starch
+Gelatin
respectively

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3.4.4. Molecular identification of bacteria
In the present study three genera have been presumptively identiﬁed as,
Deinococcus radiodurans, Shewanella oneidensis, and Bacillus thuringiensis on
the basis of morphological and biochemical tests. Also, these bacteria were
subjected to 16S rRNA gene sequence analysis.
DNA isolated from each isolate of bacteria was amplified in the presence of
universal primer 27 (5′-AGAGTTTGATCCTGGCTCAG-3′), and 1492R (5′-
GGTTACCTTGTTACGACTT-3′) for the variable regions of 16S rRNA. From Fig
(3) each isolate gave only one band at expected size, which is the same as the
band produced from positive control (Lanes 1, 2). The sequence of 16S rRNA
of bacteria was submitted to Blastn (database 16S ribosomal RNA sequences
(Bacteria and Archaea) Megablast). http: //www.ncbi.nlm.nih.gov/blast. That
indicated a close genetic relatedness of bacteria with the16S rRNA sequence of
D. radiodurans, S. oneidensis, and Bacillus thuringiensis. The highest sequence
similarity of the bacteria are as follows Bacillus (showed 99% similarity with
Bacillus thuringiensis Accession No: 346665.1), and Deinococcus (99%
similarity with Deinococcus radiodurans Accession No: AM292065.1), and
Shewanella (showed similarity 98% with Shewanella oneidensis Accession No.
AEO14299.2).

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Figure 2: PCR products (A) M, Fermentas GeneRuler 1000 bp; Lanes: 1, PCR products
from samples D; 2, PCR products from samples S.; N, negative control.(B) M, Fermentas
GenRuler 1000pb DNA lader Plus ; Lanes 1, PCR products from sample B. , N , negative
control.
1405 bp
1000bp
A
1402bp
1000bp
B
M 1 2 N M
M 1 N
1420bp

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DNA Sequence of Deinococcus: (1405 bp)
CGAACGCGGTCTTCGGACCGAGTGGCGCACGGGTGAGTAAAGCGTAACTGACCTACCCAGAAGTCATG
AATAACTGGCCGAAAGGTCAGCTAATACGTGATGTGATGATTCGCTTTGGCGAATCATTAAAGATTTA
TCGCTTCTGGATGGGGTTGCGTTCCATCAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCGACGACGG
ATAGCCGGCCTGAGAGGGTGGCCGGCCACAGGGGCACTGAGACACGGGTCCCACTCCTACGGGAGGCA
GCAGTTAGGAATCTTCCACAATGGGCGCAAGCTTGATGGAGCGACGCCGCGTGAGGGATGAAGGTTCT
CGGATCGTAAACCTCTGAACTAGGGACGAAAGAGCCGTATGGCAGATGACGGTACCTAGGTAATAGCA
CCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTACCCGGAATCACTGGGCG
GCGGAATGTTAAGTCTGGTTTTAAAGACTGGGGCTCAACCCCAGGAGTGGACTGGATACTGGCAATCT
TGACCTCTGGAGAGGTAACTGGAATTCCTGGTGTAGCGGTGGAATGCGTAGATACCAGGAGGAACACC
AATGGCGAAGGCAAGTTACTGGACAGAAGGTGACGCTGAGGCGCGAAAGTGTGGGGAGCAAACCGGAT
TAGATACCCGGGTAGTCCACACCCTAAACGATGTACGTTGGCTCATCGCAGGATGCTGTGATGGGCGA
AGCTAACGCGATAAACGTACCGCCTGGGAAGTACGGCCGCAAGGTTGAAACTCAAATGAATTGACGGG
GGCCCGCACAAGCGGTGGAGCATGTGGTTTACTTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGAC
ATGCTAGGAAGAGCGCAGAGATGCGCTCGTGCCCTTCGGGGAACCTAGACACAGGTGCTGCATGGCTG
TCGTCAGCTCGTGTCGTGAGATGTTGGGGTTAAGTCCCGCAACGAGCGCAACCCCTACCTTTAGTTGC
CAGCATTGAGTTGGGCACTCTAGAGGGACTGCCTATGAAAGTAGGAGGAAGGCGGGGATGACGTCTAG
TCAGCATGGTCCTTACGTCCTGGGCTACACACGTGCTACAATGGGTAGGACAACGCGCAGCAAACATG
CGAGTGTAAGCGAATCGCTGAAACCTACCCCCAGTTCAGATCGGAGTCTGCAACTCGACTCCGTGAAG
TTGGAATCGCTAGTAATCGCGGGTCAGCATACCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGC
CCGTCACACCATGGGAGTAGATTGCAGTTGAAACCGCCCGGGAGCCTCACGGCAGGCGTCTAGACTGT
GGTTTATGACTGGGGTGAAGTCGTAACAAGG
DNA Sequence of Shewanella: (1420 bp)
GAGAGTTTGATCTGGCTCAGATCTGCCCAGTCGAGTTTGATAACAGTTGGAAACGACTGCTAATAC
CGCATACGCCCTACGGGGGAAAGAGGGGGACTTTCGGGCCTCTCGCGATTGGATGAACCTAGGTG
GGATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGACGATCCCTAGCTGTTCTGAGAGGATG
A
TCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCA
CAATGGGGGAAACCCTGATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCAC
TAATAGTAGGGAGGAAAGGGTAANTCCTAATACGNCTTATCTGTGACGTTACCTACAGAAGAAGG
ACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTCCNAGCGTTAATCGGAATTACTG
G
GCGTAAAGCGTGCGCAGGCGGTTTGTTAAGCGAGATGTGAAAGCCCTGGGCTCAACCTAGGAATC
GCATTTCGAACTGACCAACTAGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAAT
G
CGTAGAGATCTATGGTACTACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCATGCA
CGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCTACTC
GGAGTTTGGTGTCTTGAACACTGGGCAAGCAAGCTAACGCATTAAGTAGACCGCCTGGGGAGTAC
GGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTA
ATTCGATGCAACGCGAAGAACCTTACCTACTCTTGTTTCACGGAAGACTGCAGAGATGCGGTTGTG
CCTTCGGGAACCGTGAGACAGGTGCTGCATGGCTGTCGTATGCTCGTGTTGTGAAATGTTGGGTT
AAGTCCCGCAACGAGCGCAACCCCTATCCTTATTTGCCAGCACGTGGAGGTGGGAACTCTAGGGA
G
ACTGCCGGTGATAAACCGGAGGAAGGTGGGGACGACGTCAATTCATCATGGCCCTTACGAGTAGG
GCTACACACGTGCTACAATGGCGAGTACAGAGGGTTGCAAAGCCGCGAGGTGGAGCTAATCTCAC
AAAGCTCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCCCTAGTAATC
GTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGG
A
GTGGGCTGCAAAAGAAGTGGGTAGCTTAACCTTCGGGGGGGCGCTCACCACTTTGTGGTTCATGAC
TGGGGTGAAGTCGTAACTTCCTCCACAGGTGAAGGTAGCCGTAAAT
DNA Sequence of Bacillus: (1402 bp)

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AGTCGAGCGAATGGATTAAGAGCTTGCTCTTATGAAGTTAGCGGCGGACGGGTGAGTAACACGTG
GGTAACCTGCCCATAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATAACATTTTGA
ACCGCATGGTTCGAAATTGAAAGGCGGCTTCGGCTGTCACTTATGGATGGACCCGCGTCGCATTAG
CTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCC
ACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGG
ACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGT
TAGGGAAGAACAAGTGCTAGTTGAATAAGCTGGCACCTTGACGGTACCTAACCAGAAAGCCACGG
CTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGT
AAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGGTCAT
TGGAAACTGGGAGACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGT
AGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACACTGAGGCGCGA
AACGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTG
TTAGAGGGTTTCCGCCCTTTAGTGCTGAAGTTAACGCATTAAGCACTCCGCCTGGGGAGTACGGCC
GCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTC
GAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAACCCTAGAGATAGGGCTTCTCC
TTCGGGAGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAG
TCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCATCATTAAGTTGGGCACTCTAAGGTGACTGC
CGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTAC
ACACGTGCTACAATGGACGGTACAAAGAGCTGCAAGACCGCGAGGTGGAGCTAATCTCATAAAAC
CGTTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCTGGAATCGCTAGTAATCGCGGA
TCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTG
TAACACCCGAAGTCGGTGGGGTAACCTTT
3.5. Minimum Inhibitory Concentration (MIC)
The MIC is the lowest concentration of the heavy metals that completely
inhibited bacterial growth. From table (11) B. thuringiensis exhibited lower
MIC for Cd (50 mg/l), while it has higher MIC for Pb (1800 mg/l). S.
oneidensis appeared higher MIC for Cd (1000 mg/l), and its MIC for Pb was
(700 mg/l). MIC for Cd and Pb were 600 and 400 respectively in D.
radiodurans. (Pic.10). The variance of analysis of data shows significant
(P0.05) differences in MIC for both metals (Pb and Cd) in related to studied
bacteria, as it was clear from LSD value (294.50 and 394.00) respectively.
Table 11: Minimum inhibitory concentration of Cd and Pb by D. radiodurans,
S. oneidensis and B. thuringiensis.
MIC (mg/l)Species
CdPb
600400D. radiodurans
1000700S. oneidensis
501800B. thuringiensis

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53
C
A B
D
Picture 10: MIC experiment A and B, represent the sensitivity of the bacteria
to Pb and Cd. C and D represent resistance the of bacteria to Pb
and Cd.

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54
3.6. Bioaccumulation study
3.6.1. Deinococcus radiodurans
From the results show in Fig. 3, there is an increasing in the accumulation
of Pb by D. radiodurans with increasing the concentration. The highest
accumulation occurs at the concentration 50 mg/l after 6h of incubation
(0.33mg/g), while the lowest was 0.029 mg/g after 2h in concentration 5 mg/l.
In addition, the rising of incubation period from 2 to 4 and to 6 h increase the
accumulation rate, but the incubation period (24 and 48 h) reduces the
accumulation for all concentrations. The analysis of variance of
bioaccumulation of Pb and Cd between time and concentration was significant
(P0.05) in all treatments from LSD value (0.0049).
The accumulation of Cd by D. radiodurans (Fig. 4) increased parallel with
the increasing of concentration for all the incubation period. The figure clarifies
increasing in Cd accumulation with time (2, 4 and 6 h.) for concentration (10,
20 and 50 mg/l), then decrease during (24 and 48h). For concentration
(100mg/l), accumulation increase with time (2, 4, 6 and 24h), then decrease
with time (48h). The analysis of variance of bioaccumulation of Pb and Cd
between time and concentration was significant (P0.05) in all treatments from
LSD value (0.0341).
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
Bioaccumulation(mg/g)
2 4 6 24 48
Time( hr)
Bioaccumulation of Pb
con 5
con10
con 25
con 50
control
Figure 3: Bioaccumulation (mg/g) of Pb by D. radiodurans at different

$*-!+--0)
55
0
5
10
15
20
25
30
35
40
2 4 6 24 48
Time( hr)
Bioaccumulation of Cd
con.10
con. 20
con. 50
con. 100
control
Figure 4: Bioaccumulation (mg/g) of Cd by D. radiodurans at different
To understand the manner of Pb accumulation by S. oneidensis. Fig (5),
shows that the Pb accumulation increases with the increases of both of
concentration and incubation period time. The highest accumulation was
3.98mg/g at concentration 50 mg/l at 48 h, while the lowest was 0.10mg/g at
concentration 5 mg/l at 2 h. For Cd Fig. (6), shows increased in accumulation
with the increase of concentration and time, but the increase with time was
considerable in comparison with that for the time. The maximum accumulation
occurs in the concentration 100 mg/l at 48 h (26.77 mg/g). Finally, from figures
(6 and 7) it is clear that the ability of S. oneidensis to accumulate Cd raises in
comparison with its ability to accumulate Pb. Statistical analysis of variance of
bioaccumulation of Pb and Cd between time and concentration was significant
(P0.05) in all treatments as seen from LSD value (0.0049 and 0.024)
respectivelly.

$*-!+--0)
56
0
0.5
1
1.5
2
2.5
3
3.5
4Bioaccumulation(mg/g)
2 4 6 24 48
Time(hr)
con. 5
con. 10
con. 25
con 50
control
Figure 5: Bioaccumulation (mg/g) of Pb by S. oneidensis during different
Figure 6: Bioaccumulation (mg/g) of Cd by S. oneidensis during different
3.6.3. Bacillus thuringiensis
The potentiality of Pb accumulation by B. thuringiensis has been illustrated
in Fig (7). The accumulation ability of this bacterium changes with the change
of incubation period and concentrations. So, the highest accumulation was
11.95 mg/g at concentration 50mg/l for24h, while the lowest was 1.17mg/g at
concentration 5mg/l for 2 h. The analysis of variance of bioaccumulation of Pb
0
5
10
15
20
25
30
2 4 6 24 48
Time( hr)
Bioaccumulation Cd
con 10
con20
con 50
con 100
control

$*-!+--0)
57
and Cd between time and concentration was significant (P0.05) in all
treatments from LSD value (0.0049).
Fig(8)Shows the accumulation rate of Cd by B thuringiensis. The
accumulation increased with the increase of both of incubation period and
concentrations. The highest accumulation was 22.70 mg/g at concentration
100mg/l for 48h.The lowest was 2.50mg/g at concentration 10mg/l for 2h. The
analysis of variance of bioaccumulation of Pb and Cd between time and
concentration was significant (P0.05) in all treatments from LSD value
(0.0341).
0
2
4
6
8
10
12
Bioaccumulation(mg/g
)
1 2 3 4 5
Time(hr)
5
10
25
50
control
Figure 7: Bioaccumulation (mg/g) of Pb by B. thuringiensis during different
0
5
10
15
20
25
Bioaccumulation
(mg/g)
2 4 6 24 48
Time(hr)
Bioaccumulation of Cd
con 10
con 20
con 50
con 100
control
Figure 8: Bioaccumulation (mg/g) of Cd by B. thuringiensis during different

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58
3.7 Biosorption study
3.7.1. Deinococuus radiodurans
Table (12) shows the ability of living cells of D. radiodurans to absorb both
Pb and Cd at different period of incubation and different concentrations. From
the table, the absorption of Pb increased with different periods of incubation for
the concentrations (5, 10, 25 mg/l). While it was decreased with the incubation
period for the concentration 50 mg/l. The high absorption obtained was 63.46 %
in concentration 50 mg/l at 2h, while the low was 22.72 % at concentration 5
mg/l for 2h.
The absorption of Cd decreased with the period of incubation. The high
absorption was 31.23% at concentration 50 mg/l for 2h of incubation, while the
low absorption was 15.58% for concentration 5 mg/l for 48h of incubation
(Table12). The variance of analysis of data shows significant (P0.05)
differences in biosorption for both metals in related to time and concentration,
as it was clear from LSD value.

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59
Table 12: Biosorption (%) of Pb and Cd at different period of incubations and
different concentrations by D. radiodurans.
%Biosorption of Pb at different incubation period
(h.).
Concentration
(mg/l)
4824642
30.4327.9123.5022.8522.725
30.4530.4330.4030.3228.1510
44.3344.2044.0043.2041.825
31.8933.4039.3359.9763.4650
LSD= 0.001
%Biosorption of Cd at different incubation period
(h.).
Concentration
(mg/l)
4824642
15.5816.1017.6518.5718.975
20.1420.7121.0022.7622.8010
26.5226.5727.5827.6027.6425
30.3230.6430.7531.1231.2350
LSD= 0.024
From Table (13) the biosorption of Pb by S. oneidensis decreased with the
increasing of the incubation period for all concentrations. The highest
absorption was 51.06% at concentration 50 mg/l for 2h of incubation, while the
lowest absorption was 14.00% at concentration 5 mg/l for 48h of incubation.
The biosorption of Cd has the same approach of Pb, in which the absorption
decreased with the increasing of the incubation period. The highest absorption

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60
was 42.64% at concentration 5 mg/l, for 2h of incubation and the lowest was
10.41% at concentration 50 mg/l, for 48h of incubation.
Statistically the data show significant (P0.05) differences in biosorption for
both metals in related to time and concentration.
Table 13: Biosorption (%) of Pb and Cd at different incubation period and
different concentrations by S. oneidensis.
%Biosorption of Pb at different incubation period
(h).
Concentration
(mg/l)
4824642
14.0014.7815.0816.3019.525
31.0031.8532.5233.1834.5110
39.2340.5243.4044.4248.2325
42.1143.0644.4048.1351.0650
LSD=0.001
%Biosorption of Cd at different incubation period
(h).
Concentration
(mg/l)
4824642
25.5828.2032.4141.0742.645
15.215.6316.7822.3727.2010
12.0212.2212.7414.5020.6825
10.4110.9911.5511.6514.8750
LSD=0.024

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61
3.7.3. Bacillus thuringiensis
From Table (14) the biosorption of Pb decreased with the increasing of
incubations period for all concentrations. The highest absorption was 69.64% at
concentration 50 mg/l for 2h of incubation, while the lowest absorption was
15.28% at concentration 10 mg/l for 48h of incubation.
The biosorption of Cd also decreased with the increasing of the incubation
period. The highest absorption was 93.06% at a concentration 5mg/l for 2h of
incubation and the lowest was 5.00% at concentration 5 mg/l, for 48h of
incubation. There is a significant difference (P0.05) between all concentrations
and times.
Table 14: Biosorption (%) of Pb and Cd at different incubation period and
different concentrations by B. thuringiensis.
% Biosorption of Pb at different incubation period (h).
Concentration
(mg/l)
4824642
19.0520.1220.6920.7021.365
15.2815.3815.4015.7319.9310
53.0058.3559.3861.9262.8025
48.3355.4257.1867.5769.6450
LSD=0.001
%Biosorption of Cd at different incubation period (h).
Concentration
(mg/l)
4824642
5.005.556.006.406.595
40.1240.1840.8236.9337.2210
76.3275.4773.4673.6373.7325
91.6691.7892.7992.9093.0650
LSD= 0.024

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