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Isolation and characterization of Cd-resistant bacteria
from industrial wastewater
Syed Zaghum Abbasa, Mohd. Rafatullaha, Norli Ismaila & Japareng Lalunga
a Division of Environmental Technology, School of Industrial Technology, Universiti Sains
Malaysia, Penang 11800, Malaysia, Tel. +604 653 2111; Fax: +604 657 3678
Published online: 23 Jul 2014.
To cite this article: Syed Zaghum Abbas, Mohd. Rafatullah, Norli Ismail & Japareng Lalung (2014): Isolation and
characterization of Cd-resistant bacteria from industrial wastewater, Desalination and Water Treatment, DOI:
10.1080/19443994.2014.941304
To link to this article: http://dx.doi.org/10.1080/19443994.2014.941304
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Isolation and characterization of Cd-resistant bacteria from industrial
wastewater
Syed Zaghum Abbas, Mohd. Rafatullah*, Norli Ismail, Japareng Lalung
Division of Environmental Technology, School of Industrial Technology, Universiti Sains Malaysia, Penang 11800, Malaysia,
Tel. +604 653 2111; Fax: +604 657 3678; emails: zaghum2009@yahoo.com (S.Z. Abbas), mohd_rafatullah@yahoo.co.in,
mrafatullah@usm.my (M. Rafatullah), norlii@usm.my (N. Ismail), japareng@usm.my (J. Lalung)
Received 7 January 2014; Accepted 27 June 2014
ABSTRACT
Three bacterial strains were isolated from industrial effluents of Penang, Malaysia. The
strains RZ1, RZ2, and RZ3 were identified as Pantoea sp. RL32.2, Salmonella enterica, and
Enterobacter sp. OCPSB1, respectively, based on morphological observation, biochemical,
physiological characterization, and 16S rDNA sequence analysis. The strains RZ1, RZ2, and
RZ3 removed 89.89, 82.10, and 89.14% of cadmium, respectively, when the 100 μg/mL of
cadmium was added in the medium. The minimum inhibitory concentrations of strains
RZ1, RZ2, and RZ3 were 750, 410, and 550 μg/mL, respectively. Cured strain showed resis-
tance and sensitivity against some range of antibiotics. The molecular weights of induced
proteins were 35 and 25 kDa in the presence of cadmium which points out a possible role
of this protein in cadmium removal. Overall, these strains could be useful for the removal
of cadmium in industrial wastewater.
Keywords: Absorbance; Antibiotics; Bioremediation; Cadmium; Isolation
1. Introduction
Cadmium (Cd) is a heavy metal contaminant in
the environment. Extensive data suggest Cd is the
most toxic heavy metal and it is included in the black
list of several international agreements established to
regulate the input of Cd into the environment [1]. It is
extensively used in the industry for a number of
applications, including electroplating, protection
against corrosion, and stabilizing plastic [2]. Wastewa-
ters of these industries contain Cd ranging from 10 to
100 mM [3]. Cd can enter the human food chain
through plants, smoking materials, and diet [4]. Cd is
carcinogenic, embryotoxic, teratogenic, and mutagenic
and may cause hyperglycemia, reduced immunopo-
tency, and anemia, due to its interference with iron
metabolism [5]. The toxicity of Cd has also been well
documented in selective types of almost all major
phyla of eukaryotes [6].
Conventional methods such as precipitation, oxida-
tion, or reduction have been commonly used to
remove heavy metals from industrial wastewater [7].
They are ineffective and expensive. So, substitute
methods of Cd removal and revival based on biologi-
cal resources have been considered [8]. Among micro-
organisms, bacteria, yeast, and protozoa are generally
the first category to be exposed to Cd present in the
environment [9]. The Cd-resistant bacteria have been
isolated in a number of studies which explain the
mechanism of Cd uptake in bacteria [10]. Moreover, it
*Corresponding authors.
1944-3994/1944-3986 Ó2014 Balaban Desalination Publications. All rights reserved.
Desalination and Water Treatment (2014) 1–10
www.deswater.com
doi: 10.1080/19443994.2014.941304
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is also indicated that Gram-negative bacteria are
highly resistant to Cd ions and metabolize great
amounts of Cd during growth. Gram-positive bacteria
on the other hand, show a heterogeneous behavior
and the intracellular pool reaches a plateau on increas-
ing Cd concentration [11].
Bacterial genes that are involved in Cd-resistant
are located on plasmids that harbor many genes
against many metals [12]. In Cd-resistant bacteria four
different mechanisms are present to resist the Cd. First
one is efflux mechanism in which CadA and CadB
genes are involved and they resist against zinc (Zn)
and Cd. Second mechanism is based on enzymes that
make the bacterial cell wall impermeable to Cd in
which manganese transport system and zinc transport
system are involved. Both systems are coded by chro-
mosome. Third mechanism is the conversion of Cd
into non-toxic form of enzymes. After bioconversion,
the Cd undergoes valency change as a result less toxic
and volatile compounds have been produced in many
cases. Fourth, Cd-resistant bacteria have developed
mechanisms to bind Cd to surface factor or intracellu-
lar binding. A mutant Citrobacteri isolated from
metal-polluted area accumulates Cd inside the cell as
insoluble cell-bound CdHPO
4
during the growth in
the presence of glycerol 2-phosphate and Cd. The sur-
face of the cell is most important for the precipitation
of metal [13]. In some cases, Cd has been shown to
bind to the capsular surface in Klebsiella aerogenes and
in Arthrobacter viscosus [14]. Binding of Cd by thiols:
this mechanism is important in the eukaryotic organ-
isms but in case of bacteria, polythiols Cd binding
peptide is analogous to metallothionein of animal cells
[15].
Although different Cd-resistant bacterial species
are used for the removal of Cd, the isolation of poten-
tial Cd-resistant bacteria and characterization of Cd
removal properties are still required in order to
remove the Cd contaminant from environment. The
aim of this study was to isolate bacterial strains with
high Cd-resistance. The physiological, biochemical,
and molecular features were used to characterize the
strains. Phylogenetic analysis, based on 16S rDNA
gene sequence data, was also used to reveal the
genetic relationship between the strains and others.
The protein-profiling technique was also used to char-
acterize strains on protein level.
2. Materials and methods
2.1. Chemicals
Lauria bertani (LB) broth and LB agar were pur-
chased from Hi-media laboratories (India), cadmium
chloride (CdCl
2
) was procured from Sigma Aldrich
(USA), penicillin (10 IU), tetracycline (30 μg), amoxicil-
lin (10 μg), gentamycin (10 μg), cephalexin (30 μg),
erythromycin (15 μg), streptomycin (10 μg), ciprofloxa-
cin (5 μg), and M9 acetate minimal medium (0.5 g/mL
yeast extract, 0.2 g/mL MgSO
4
, 5.0 g/mL sodium ace-
tate, 0.001 g/mL FeSO
4
, 0.001 g/mL CaCl
2
(as agglom-
erating agent), 0.5 g/mL K
2
HPO
4
(potassium and
phosphorous source), and 1.0 g/mL NH
4
Cl).
2.2. Sampling
Wastewater effluent samples of Globetronics
Industries Sdn. Bhd. and Ever-prosper Battery & Tyres
Sdn. Bhd. Penang, Malaysia were collected from Ba-
yan Lepas zone/area located at about 1 km away from
the source of wastewater effluent. A total of four sam-
ples were taken from both localities in sterilized
screw-capped bottles and brought to the microbiology
laboratory of Universiti Sains Malaysia. The parame-
ters such as pH, temperature, longitude, latitude, and
Cd concentration were noted.
2.3. Isolation of Cd-resistant bacteria
LB agar plates (pH adjusted to 7 ± 7.5) containing
CdCl
2
with Cd concentration 10 μg/mL were used to
select Cd-resistant bacteria and 20 μL of each wastewa-
ter sample was spread on agar plates and incubates at
37˚C for 24 h [16]. Bacterial colonies growing on agar
plates were purified by streaking and re-streaking.
2.4. Identification of bacterial isolates based on classical
taxonomy and 16S rDNA
Among the selected bacterial strains which can
remove Cd, the morphological, physiological, and bio-
chemical characteristics were evaluated. For morpho-
logical and physiological characterization like colony
color was observed under 10X lens, shape and nature
of strains either Gram-negative or Gram-positive were
observed under light microscope after Gram’s stain-
ing. The ability of bacterial isolates of spore forming
or non-spore forming was checked by endospore
staining. The motility of isolated strains was checked
by motility test. In this test, semi-solid agar was pre-
pared in the test tubes and stabbed by inoculating
needle that was already charged with bacterial culture.
These test tubes were incubated at 37˚C for 24 h. Taxo-
nomic identification of those isolates was carried out
using Bergey’s Manual of Determinative Bacteriology.
Confirmation of the taxonomical status of the selected
strains was done by molecular methods. For further
2S.Z. Abbas et al. / Desalination and Water Treatment
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identification, the genomic DNA was extracted using
Sigma’s GenElute Bacterial Genomic Kit according to
the manufacturer’s instructions and the 16S rDNA
gene was amplified by polymerase chain reaction
(PCR) using the universal 16S rDNA primers 27F
(5´AGAGTTTGATCMTGGCTCAG 3´) and 1492R
(5´TACGGYTACCTTGTTACGACTT 3´) [17]. The reac-
tion conditions were as follows: 95˚C for 3 min; 32
cycles: 94˚C for 1 min, 56˚C for 1 min and 72˚C for
2 min; 72˚C for 10 min and 4˚C pause. PCR products
corresponding to the expected size of amplified 16S
rDNA (1.5 kb) were purified with a Gel Extraction Kit
(Invitrogen) and sent to Center of Chemical Biology,
Universiti Sains Malaysia Co. Ltd. for sequencing.
2.5. Phylogenetic analysis
16S rDNA sequence was submitted to the database
of GenBank and compared with similar sequences by
BLAST analysis. Phylogenetic trees of 16S rDNA were
constructed using the software Clustal W [3].
2.6. Cd removal
The 20 μL of bacterial culture was added in each
flask which consisted of 100 μg Cd/mL. Subsequently,
these flasks were placed on shaking incubator at 37˚C
and aliquots (1 mL) were taken after every 4 h inter-
vals up to 24 h for Cd estimation [18]. Each time the
cultures were spun using centrifuge machine (Hermle,
Ind Co. Ltd. China) at 6,000 rpm/min to remove bacte-
rial cells, and the supernatant was used to determine
Cd concentration by GBC932 atomic absorption spec-
trophotometry (Pantech Instruments, Blackburn, Victo-
ria, Australia) at 228.8 nm using a Cd lamp. The
amount of Cd in samples after various intervals of
time was estimated using a standard curve, which
was prepared by taking various known concentrations
of Cd in the medium. Reduction in the amount of Cd
in the medium after growth of bacteria was taken as
the Cd uptake ability of the isolates.
2.7. Effect of pH and temperature on the removal of Cd
To see the effect of pH and temperature on the Cd
removal the bacterial isolates. The 50 mL LB broth was
taken in each conical flask to test the effect of different
pH (1.0–13.0) and temperatures (5–45˚C) on Cd
removal by bacterial isolates. These flasks were inocu-
lated with 20 μL of bacterial culture followed by incu-
bation at the desired temperatures and adjusted with
particular pH. These experiments were performed in
triplicates. At each pH and temperature, the bacterial
cells and supernatant were separated to check the Cd
concentration by using atomic absorption spectropho-
tometry at 228.8 nm using a Cd lamp.
2.8. Determination of optimum growth conditions
For optimum growth of the bacterial isolates, two
parameters, i.e. temperature and pH, were considered
in both control and Cd-stressed conditions. For each
bacterial isolate, 5 mL LB broth was added into 18
sets, each set consisted of three test tubes. The tubes
were autoclaved and inoculated with 20 μL of the
freshly prepared culture of each bacterial isolate
grown over night at 37˚C. The nine sets of tubes with-
out Cd-stress and nine sets of tubes with Cd-stress
(100 μg/mL) were incubated at 5, 10, 15, 20, 25, 30, 35,
40, and 45˚C, respectively. After an incubation period
of 12 h, their absorbance was measured at 600 nm
using a LAMBDA 650 UV/vis spectrophotometer
(Perkin Elmer, New York, USA). To determine the
optimum pH, test tubes having 5 mL LB broth was
prepared in 12 sets, each containing 3 test tubes and
their pH was adjusted to 1.0, 3.0, 5.0, 7.0, 9.0, and 13.0
and then autoclaved. These tubes were inoculated
with 20 μL freshly prepared cultures of each bacterial
isolate. The six sets of test tubes were incubated with
Cd-stress (100 μg/mL) and six sets of test tubes were
incubated without Cd-stress. After an incubation per-
iod of 12 h, their absorbance was measured at 600 nm.
2.9. Effect of Cd on bacterial growth
The growth curves of bacterial isolates were deter-
mined in LB broth with 100 μg/mL of Cd and in con-
trol medium. For each bacterial isolate, 50 mL medium
was taken in one set consisting of three flasks, auto-
claved and then inoculated with 20 μL of the freshly
prepared inoculums. The cultures were incubated at
37˚C in a shaker at 80–100 rotation/min. An aliquot of
culture was taken out in an oven-sterilized tube at
regular intervals of 0, 4, 8, 12, 16, 20, 24, and 32 h.
Absorbance was measured at 600 nm and growth was
plotted graphically.
2.10. Determination of Cd-tolerance
The growth measurement is used to evaluate
the resistant properties of bacterial strains. The Cd-
resistance of bacterial isolates was determined using
stock solutions of 10 μg/mL of CdCl
2
. The Cd-resistance
was checked by increasing the concentration of
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respective Cd in a stepwise manner with 50 μg/mL of
Cd-checked resistance in the M9 acetate minimal
medium Culture flasks containing 100 mL of medium
and Cd
2+
ions were inoculated with 20 μL overnight
bacterial cultures and incubated at 37˚C for 24 h.
Growth was measured as optical density at 600 nm.
2.11. Determination of antibiotic resistance
These strains were tested for their sensitivity
against eight antibiotics that were given above. The
0.1 mL bacterial culture was plated onto LB agar
plates. The antibiotic disks were placed on the plates
and incubated at 37˚C for 1–2 d.
2.12. Protein profiling
In conical flasks, 20 mL LB broth was taken in trip-
licates and steam sterilized. Bacterial isolates were
stressed with concentrations of Cd 350 μg/mL with
control and were incubated for 16 h at 37˚C in shaking
incubator and cells were harvested by centrifugation.
The pellet was dissolved in 100 μL of 1X loading dye
and then heat shock was given for 5 min, eppendorf
was shifted on ice for 2 min and then was centrifuged
at 12,000 rpm for 10 min. Supernatant was transferred
to a new eppendorf, then the final centrifugation was
done at 12,000 rpm for 10 min, and the supernatant
was shifted to a new eppendorf. The bacterial proteins
were resolved by SDS-PAGE. Electrophoresis was
performed in vertical mini-slab gel (Mini-Protean III;
BioRad) with a gel thickness of 0.75 mm and gel size
8×7 cm. The gels were composed of 10% resolving gel
and 4% stacking gel and run at constant voltage of
200 V for 50 min. Amounts of 10 μL of bacterial protein
extracts were loaded into each well of the gel. After
electrophoretic separation, the gel was stained with
Coomassie blue solution (0.01% Coomassie brilliant
blue R250, 45% (v/v) methanol and 10% (v/v) glacial
acetic acid) for 30 min at room temperature and
subsequently placed in the destaining solution (50%
(v/v) methanol and 2% (v/v) acetic acid) for 1 h. The
gel image was captured and analyzed using VersaDoc
Imaging System (BioRad) [19].
3. Results and discussion
3.1. Sampling and isolation of Cd-resistant bacteria
The parameters of industrial wastewater samples
were noted as shown in Table 1. On the basis of mor-
phology and color, three Cd-resistant bacterial strains
were isolated and named as strains RZ1, RZ2, and
RZ3.
3.2. Taxonomical identification and 16S rDNA
All the selected bacterial strains RZ1, RZ2, and
RZ3 were morphologically, physiologically, and bio-
chemically characterized. The investigated bacteria
form mostly round colonies onto the agar surface. The
colors of colonies vary from off-white to white and
were slightly yellow. The cells of the isolated bacteria
were mostly cocci shape. Most of the part of the iso-
lates were Gram-negative, non-spore-forming bacteria
and mostly were motile. After physiologo-biochemical
tests, that were carried out, it comes to the next more
important characteristics of the tested bacteria as
shown in Table 2. All three strains could not degrade
the hydrogen peroxide and urea and were also unable
to hydrolyze gelatin. The strain RZ1 failed to ferment
carbohydrate but strains RZ2 and RZ3 were able to
ferment carbohydrate. Both strains RZ1 and RZ2
showed negative on MRVP test but strain RZ3 showed
positive results for mixed acid fermentation. The
strains RZI and RZ2 were unable to use citrate as a
carbon source while strain RZ3 was metabolized eas-
ily. All the three strains were non-pathogenic, fastidi-
ous, and lactose fermenting. The taxonomic status of
these isolates was determined using Bergey’s Manual
of Determinative Bacteriology [20], and showed that
Table 1
Collection of water samples
S. no. Industries Samples pH Temp. (˚C) Longitude Latitude
Cadmium
concentration (mg/L)
1 Globetronics Industries Sdn. Bhd. Sample 1 6.0 30 5˚.07617´N 100˚.386119´E 0.36
Sample 2 7.0 29 5˚.38400´N 100˚.302104´E 0.52
2 Ever-prosper Battery & Tyres Sdn. Bhd. Sample 3 6.0 35 5˚.41264´N 100˚.325307´E 0.67
Sample 4 7.0 33 5˚.329379´N 100˚.482284´E 0.82
4S.Z. Abbas et al. / Desalination and Water Treatment
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strains RZ1, RZ2, and RZ3 belong to genus Pantoea,
Salmonella, and Enterobacter, respectively.
“Universal” eubacterial primers were used to
amplify 1.5 kb of 16S rDNA of the strains RZ1, RZ2,
and RZ3. The PCR products obtained were purified
and sequenced by using BLAST to compare its
sequence to all sequences in the NCBI database
(http://www.ncbi.nlm.nih.gov). The 16S rDNA genes
of strains RZ1, RZ2, and RZ3 were 97% homologous
to Pantoea sp. RL32.2 (Accession number: GU056355),
97% homologous to Salmonella enterica (Accession
number: KF056927), and 95% homologous to Entero-
bacter sp. OCPSB1 (Accession number: JN119828),
respectively.
3.3. Phylogenetic analysis of 16S rDNA
Nucleotide sequence analysis of the 16S rRNA gene
has been considered a fast and accurate method to iden-
tify the phylogenetic position of bacterial strains.
The full-length 16S rDNA of strains RZ1, RZ2, and RZ3
were sequenced and used to construct phylogenetic
development trees as shown in Fig. 1. We found that
strain RZ1 was classified in the branch of Pantoea sp. It
has 97% similarity with Pantoea sp. RL32.2 as shown in
Fig. 1(a). The RZ2 strain has 97% similarity with
S. enterica subsp. Entericaserovar typhi, which is classified
under the family of Enterobacteriaceae 1736 as shown
in Fig. 1(b). The strain RZ3 was classified under
Enterobacter, it has 95% similarity with Enterobacter sp.
OCPSB.
3.4. Cd estimation
These results showed that all the three strains were
efficient in the removal of Cd as shown in Fig. 2. The
three different strains showed different efficiency in
removing Cd. The strains RZ1 and RZ2 were more
efficient during the late log phase and less efficient
during the early log phase, while strain RZ3 was less
efficient in the late log phase and more efficient in the
early log phase. Within 4 h of inoculation, strain RZ3
removed 52% while 8% removal in the presence of
strain RZ1 and 10% removal in the presence of strain
RZ2. After 8 h of incubation, strains RZ1 and RZ2
showed 58 and 40% reduction in the Cd, respectively,
while strain RZ3 showed only 1% Cd removal. After
24 h, this reduction ranged between 82 and 89% in the
three strains. Generally, the process of micro-organism
removal of heavy metals includes biosorption and bio-
accumulation. Biosorption process means metal ions
are adsorbed by micro-organisms through the bio-
chemical reactions including complex, chelate, ions
exchange, and adsorption. Zeng et al. [3] described
that Pseudomonas aeruginosa strain E1 uses these two
mechanisms for the removal of Cd from industrial
wastewater. Both living and non-living cells have bio-
sorption. Bioaccumulation happens only in living cells.
It is an active process and needs energy provided by
the metabolism of micro-organisms [21].
3.5. Effect of pH and temperature on Cd removal
Maximum removal of Cd was observed 89.89,
82.10, and 89.14% by strains RZ1, RZ2, and RZ3 at pH
7.0 because pH 7.0 was the optimum pH of these bac-
terial strains, and at low and high pH no Cd removal
was noted as shown in Figs. 3–5. The medium pH
affects the solubility of metals and the ionization state
of the functional groups (carboxylate, phosphate, and
amino groups) of the microbial cell [22]. The Cd
removal was decreased at higher and lower than opti-
mum temperature 35˚C because at optimum tempera-
ture the biomass of bacteria was high as shown in
Figs. 4and 6. Bioaccumulation of Cd
2+
ions decreased
with increasing temperature. At low temperatures, the
binding of heavy metal ions to micro-organism is by
passive uptake [23]. Similar conclusions were also
found by El-Deeb [24] and Mohamed [25]. The ther-
mal-resistant bacteria effectively removed the Cd due
to the production of enzymes like aldolase, RNA, and
DNA polymerases which can perform their normal
metabolism at the extremely higher temperature.
Chang et al. [26] isolated Vibrio parahaemolyticus which
showed higher thermal stability and survived in the
Table 2
Morphological and biochemical characteristics of bacterial
isolates
RZ1 RZ2 RZ3
Morphological
Colony color Off-white Yellow White
Gram nature Negative Negative Negative
Cell morphology Cocci Cocci Cocci
Motility Positive Positive Positive
Colony shape Round Round Round
Spore formation Negative Negative Negative
Biochemical tests
Catalase test Negative Negative Negative
Urease test Negative Negative Negative
Gelatin hydrolysis test Negative Negative Negative
Carbohydrate test Negative Positive Positive
MRVP test Negative Negative Positive
Citrate test Negative Negative Positive
Blood agar test Positive Positive Positive
Chocolate agar test Positive Positive Positive
MacConkey agar test Positive Positive Positive
S.Z. Abbas et al. / Desalination and Water Treatment 5
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range of 0–70˚C and also removed 85% of Cd from
wastewater.
3.6. Bacterial growth patterns against Cd
The growth patterns of these bacterial strains were
not significantly different from those of controls, but
the growth rate was lower in the presence of Cd
2+
ions. The growth patterns were shown in Fig. 7. This
could be explained by the toxicity of Cd, which causes
retardation in the growth of the strains due to less
enzymatic activities. Similar conclusions were reached
by Filali et al. [27].
3.7. Cd-resistance
The strain RZ1 was found resistant against Cd up
to a concentration of 750 μg/mL, but strain RZ2 can
tolerate the Cd-stress up to 410 μg/mL and resistance
level of strain RZ3 was 550 μg/mL. A common
property of many bacteria capable of growth in the
presence of Cd is their ability to prevent accumulation
Fig. 1. Phylogenetic development trees based on 16S rDNA analysis: (a) strain RZ1, (b) strain RZ2 and (c) strain RZ3.
6S.Z. Abbas et al. / Desalination and Water Treatment
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of free intracellular metal. At least two alternative
mechanisms are possible. One is that the membrane of
resistant bacteria acts as an impermeable barrier for
toxic metals while allowing the passage of physiologi-
cal cations. Cobbett and Goldsbrough [28] described
such differences in spheroplasts prepared from
Cd-sensitive or Cd-resistant strains of S. aureus.He
suggested that exclusion of toxic ions was dependent
upon conformational changes in the membrane.
Alternatively, specific sites for the continuous efflux of
Cd may operate and hence minimize the intracellular
Cd concentration. Such a system operated in Cd-resis-
tant S. aureus cells, since only the resistant strain eff-
luxed the metal when cells were transferred from a
medium containing Cd to a Cd-free medium [29].
Based on our results, the possible mechanism existed
in these strains was accumulation of Cd
2+
ions inside
bacterial cells, and formed CdHPO
4
. The formation of
CdHPO
4
occurred during the growth of bacteria in the
presence of glycerol 2-phosphate and Cd. This was
confirmed by the decrease in the concentration of Cd
at different time intervals in the medium.
0 4 8 1216202428
0
10
20
30
40
50
60
70
80
90
100
110
120
% Cd contents in the medium
Time (hours)
Control
Strain RZ1
Strain RZ2
Strain RZ3
Fig. 2. Removal of Cd after inoculation of the three bacte-
rial isolates. The medium containing the same original
amount of Cd but without inoculation was taken as con-
trol. The initial concentration of Cd
2+
ions in the medium
was 100 μg/mL.
02468101214
0
10
20
30
40
50
60
70
80
90
100
110
120
% Cd content in the medium
pH
Control
Strain RZ1
Strain RZ2
Strain RZ3
Fig. 3. The effect of various pH on the removal of Cd
2+
ions by three bacterial isolates. The initial concentration of
Cd
2+
ions in the medium was 100 μg/mL.
0 5 10 15 20 25 30 35 40 45 50
0
10
20
30
40
50
60
70
80
90
100
110
120
% Cd content in the medium
Temperature (oC)
Control
Strain RZ1
Strain RZ2
Strain RZ3
Fig. 4. The effect of various temperatures on the removal
of Cd
2+
ions by three bacterial isolates. The initial concen-
tration of Cd
2+
ions in the medium was 100 μg/mL.
02468101214
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Absorbance (at 600 nm)
pH
Strain RZ1 (control)
Strain RZ1 (Cd-stress)
Strain RZ2 (control)
Strain RZ2 (Cd-stress)
Strain RZ3 (control)
Strain RZ3 (Cd-stress)
Fig. 5. The optimum pH of bacterial isolates in the pres-
ence and absence of Cd.
S.Z. Abbas et al. / Desalination and Water Treatment 7
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3.8. Antibiotic susceptibility
In order to determine the resistance to antibiotics,
the reactions of eight antibiotics were examined by the
disk diffusion method. All three strains were sensitive
against tetracycline, gentamycin, streptomycin, and
ciprofloxacin but resistant against amoxicillin, erythro-
mycin, and penicillin. Against cephalexin, strain RZ1
was resistant while strains RZ2 and RZ3 were sensi-
tive as shown in Table 3. Under environmental condi-
tions of Cd-stress, micro-organisms might have
developed various mechanisms to resist antibiotics
and tolerate metals. Microbes surviving in polluted
water usually change intrinsic biochemical and struc-
tural properties, physiological, and genetic adaptation.
According to some findings, metal resistance is associ-
ated with multiple antibiotic resistances on R plasmid
[30,31].
3.9. Protein bands
To study the protein profile of bacteria under
stressed and non-stressed conditions, total cell pro-
teins of bacteria were isolated after 16 h of Cd expo-
sure. The Cd-stress to bacteria was given after their
optical density reached at 0.3 indicating that bacteria
have entered into a log phase. The protein gel of
stress organisms indicated new protein bands in
front of 35 and 25 kDa. This indicated that the
Cd-resistance proteins in bacteria were inductive pro-
teins and they expressed only in the stressed condi-
tions as shown in Fig. 8. The nature of the 35 and
25 kDa proteins system remains unknown and may
be the result of Cd-resistance or its cause. The
35 kDa protein appeared upon adaptation to Cd and
may therefore be a protein which controls intracellu-
lar Cd concentrations. It may be a new form of a
preexisting transport protein (33 kDa protein) with
altered specificity such that Cd uptake is reduced.
Alternatively, it may be a new protein capable of
exchanging internal Cd
2+
ions for external cations
and hence preventing Cd accumulation. These results
were in agreement with SDS-PAGE analysis by
Chovanova
´et al. [32]. They studied the membrane
proteins of adapted and non-adapted bacterial cells
and found that in adapted cells, inducible proteins
were present in membrane with molecular weight of
34.5 and 25 kDa.
0 5 10 15 20 25 30 35 40 45 50
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
Absorbannce (at 600 nm)
Temperature (oc)
Strain RZ1 (control)
Strain RZ1 (Cd-stress)
Strain RZ2(control)
Strain RZ2 (Cd-stress)
Strain RZ3 (control)
Strain RZ3 (Cd-Stress)
Fig. 6. The optimum temperature of bacterial isolates with
and without Cd-stress.
0 4 8 121620242832
0.0
0.4
0.8
1.2
1.6
2.0
2.4
2.8
3.2
Absorbance (at 600 nm)
Time (hours)
Strain RZ1 (Control)
Strain RZ1 (Cd-treated)
Strain RZ2 (Control)
Strain RZ2 (Cd-treated)
Strain RZ3 (control)
Strain RZ3 (Cd-treated)
Fig. 7. The effect of Cd on the growth patterns of bacterial
isolates.
Table 3
Resistance of antibiotics by cadmium resistant bacterial
strains
Antibiotics RZ1 RZ2 RZ3
Tetracycline (30 μg) 21 mm (S) 24 mm (S) 25 mm (S)
Amoxicilin (10 μg) R R R
Penicillin (10 units) R R R
Gentamycin (10 μg) 20 mm (S) 16 mm (S) 20 mm (S)
Cephalexin (30 μg) R 14 mm (S) 16mm (S)
Erythromycin (15 μg) R R R
Streptomycin (10 μg) 17 mm (S) 13 mm (S) 15 mm (S)
Ciprofloxacin (5 μg) 30 mm (S) 35 mm (S) 17 mm (S)
Notes: S: Sensitive; R: Resistant.
8S.Z. Abbas et al. / Desalination and Water Treatment
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4. Conclusions
The isolated bacterial strains were Gram-negative,
coccus, and highly resistant to Cd. Based on the
strains characterizations and the 16S rDNA sequence
comparison, those bacterial strains were identified as
Pantoea sp. RL32.2, S. enterica, and Enterobacter sp. OC-
PSB1. They also showed excellent Cd removal ability,
i.e. 89.89, 82.10, and 89.14%, respectively. Thus, these
bacterial isolates can be exploited for the bioremedia-
tion of Cd containing wastes due to high resistance
against Cd and potential to remove the Cd from
industrial wastewater.
Acknowledgement
The authors acknowledge the research grant pro-
vided by the Universiti Sains Malaysia under the
Short Term Grant Scheme (Project No. 304/PTE-
KIND/6312008).
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