Egyptian Journal of Aquatic Research (2017) 43, 65–74

H O S T E D BY
National Institute of Oceanography and Fisheries

Egyptian Journal of Aquatic Research

http://ees.elsevier.com/ejar
www.sciencedirect.com

Isolation and biochemical characterization of

heavy-metal resistant bacteria from tannery effluent
in Chittagong city, Bangladesh: Bioremediation
viewpoint
Lolo Wal Marzan *, Mehjabeen Hossain, Sohana Akter Mina, Yasmin Akter,
A.M. Masudul Azad Chowdhury

Department of Genetic Engineering and Biotechnology, Faculty of Biological Sciences, University of Chittagong, Chittagong 4331,
Bangladesh

Received 27 June 2016; revised 21 August 2016; accepted 30 November 2016

Available online 16 January 2017

KEYWORDS Abstract Toxic, mutagenic and carcinogenic heavy metals from tannery industries cause the pol-
Gemella sp.; lution of agricultural environment and natural water sources. This study aims to isolate, investigate
Micrococcus sp.; and identify naturally occurring bacteria capable of reducing and detoxifying heavy metals (Chro-
Hafnia sp.; mium, Cadmium and Lead) from tannery effluent. Three isolates were identified up to genus level
Heavy metal resistance; based on their morphological, cultural, physiological and biochemical characteristics as Gemella
Degradation capacity; sp., Micrococcus sp. and Hafnia sp. Among them Gemella sp. and Micrococcus sp. showed resis-
Bioremediation; tance to Lead (Pb), chromium (Cr) and cadmium (Cd), where Hafnia sp. showed sensitivity to cad-
Characterization mium (Cd). All isolates showed different MICs against the above heavy metals at different levels.
Degrading potentiality was assessed using Atomic Absorption Spectrophotometer where Gemella
sp. and Micrococcus sp. showed 55.16 ± 0.06% and 36.55 ± 0.01% reduction of Pb respectively.
On the other hand, moderate degradation of Cd was shown by Gemella sp. (50.99 ± 0.01%) and
Micrococcus sp. (38.64 ± 0.06%). Heavy metals degradation capacity of Gemella sp. and Micrococ-
cus sp. might be plasmid mediated, which might be used for plasmid transformation to transfer
heavy metal accumulation capability. Therefore, identification of three bacteria for their heavy
metal resistance and biodegradation capacity might be a base study to develop the production of
potential local bioremediation agents in toxic tannery effluent treatment technology.
Ó 2016 National Institute of Oceanography and Fisheries. Hosting by Elsevier B.V. This is an open access
article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction
* Corresponding author. Fax: +880 31 2606145, +880 31 2606014.
E-mail addresses: marzan.geb@cu.ac.bd, lmarzancu@yahoo.com
(L.W. Marzan).
Air, water and land which are the essential elements of life are
Peer review under responsibility of National Institute of Oceanography
contaminated constantly due to increasing population, rapid
and Fisheries. urbanization and industrialization (Chhikara and Dhankhar,
http://dx.doi.org/10.1016/j.ejar.2016.11.002
1687-4285 Ó 2016 National Institute of Oceanography and Fisheries. Hosting by Elsevier B.V.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
66
2008). At present, the bioaccumulation of heavy metals in
environment is a major warning to human life (Yigit and
Ahmet, 2006; Hooda, 2007). Water pollution caused by indus-
trial wastage, is frequent (Ogedengbe and Akinbile, 2004) by
toxic sludge, heavy metals, and solvents as they fall into natu-
ral water sources and agricultural environment.
Heavy metals containing industrial effluent cause health
hazards to plants, animals, aquatic life and humans increasing
pressures on the flora and fauna (Robin et al., 2012). Among
industrial usage of heavy metals, tannery industries use a sig-
nificant part of it. Tannery effluent is highly polluted because
it contains imbalance suspended solids, nitrogen, conductivity,
sulfate, sulfide and chromium, copper, cadmium and man-
ganese, biological oxygen demand (BOD) and chemical oxygen
demand (COD) (Mondal et al., 2005; Zahid et al., 2006). In
Bangladesh unprocessed tannery effluents are released into
water sources (Favazzi, 2002; Verheijen et al., 1996). Conse-
quently, the elevated concentrations of some heavy metals
are found in agricultural soils located in surrounding areas
to the tannery industries which exceed the tolerable limit.
Lead and cadmium which are major contaminants found in
the environment, are extremely poisonous to human(s), ani-
mals, plants and microbes which can damage cell membranes,
alter particularity of enzymes, and destroy the structure of
DNA. This toxicity is created by the displacement of essential
metals from their native binding sites or ligand interactions
(Olaniran et al., 2013).
Chrome powder and chrome liquor are applied in tanning
industry, and are highly toxic heavy metals (Cr6+) which cause
water pollution (Sing, 1994), where a lot of (>170000 tons)
chromium wastes are released to the surroundings
(Kamaludeen et al., 2003). It causes health hazards since it can
easily enter biological cell membranes (Chaudhary et al.,
2003). Tanned skin-cut wastes (SCW) which are used to produce
feeds and fertilizers, are the direct phenomenon of chromium
toxicity (Rafiqullah et al., 2008). Hexavalent Chromium
(Cr6+) is 100–1000 times more poisonous compared to trivalent
(Cr3+) form (Gauglhofer and Bianchi, 1991). So, conversion
of Cr6+ is one of the significant mechanisms for microorganisms
that can be used for detoxification of chromium.
Lacking a single waste-water treatment facility, a notorious
and substantial ruin of the environment is another fate of con-
cern from such a pivotal industry to sustain a billion-dollar
business. An excess of such chemicals in the water and soils
is harmful for the health of the people crammed into the area
(Sunder et al., 2010). The breakthrough toward the sustainable
mitigation of this overwhelming problem is nothing but the
installation of an appropriate effluent treatment plant in every
industry in terms of efficiency, cost effectiveness, simplicity
and more importantly it should be environment friendly.
Due to lack of treatment plants and environment management
schemes in most of the tanneries in our country, raw wastes are
simply discharged into the environment, causing severe envi-
ronmental and public health troubles in particular areas.
Appropriate environmental management is needed (Hasnat
et al., 2013) to overcome this hazardous issue and tannery pro-
ductivity. Several microorganisms have developed detoxifica-
tion and respiration mechanism using heavy metals and thus
become resistant to it (Ezaka and Anyanwa, 2011). The isola-
tion and characterization of heavy metal resistant bacteria is
significant for its metal accumulation capability along with
its resistance capacity. In our study the sampling sites are the
L.W. Marzan et al.
three nearby surroundings of Madina tannery which is the lar-
gest tannery located at Jalalabad area, near Oxygen point in
Chittagong, Bangladesh. It was established in 1983, and is
renowned for manufacturing all kinds of export quality crust
and refined leather. In the surroundings of Madina tannery,
large municipal areas have been observed.
So, the present study aims to investigate the ability of natu-
ral inhabitant bacteria of tannery effluent in reducing and
detoxifying of heavy metals (Pb, Cr and Cd) at privileged con-
ditions, where objectives include – isolation of naturally occur-
ring bacteria from tannery effluent, screening of top three
isolates as the reducer of Pb, Cr and Cd, characterization of
heavy metal resistance, identification of those bacteria up to
genus and profiling of their plasmids as a fundamental research
to ensure the basis of their resistance in order to use them for
detoxification in an incorporated bioremediation scheme.
Materials and methods
Study area and collection of samples
Tannery effluent samples were collected from three agricul-
tural and residential sites beside Madina tannery (Fig. 1D,
Table 1), in labeled pre-sterilized bottles, and cold chain was
maintained during shipment to the laboratory in the Univer-
sity of Chittagong. Collected samples were preserved at 4 °C
before analysis and during experiments.
Primary screening of heavy metal resistant bacteria
For the selective screening of heavy metal resistant bacteria,
300 lg/mL of heavy metal (Lead) incorporated LB (Luria Ber-
tani) agar plates (Peptone 10.00 g/L, yeast extract, 5.00 g/L,
NaCl 5.00 g/L, dextrose anhydrate 10.00 g/L and agar
30.00 g/L: pH
7.00) were used and screened by standard pour
plate method observed at 37 °C. After 24 h of incubation the
plates were observed for any kind of development on the cul-
ture medium. After preliminary screening of effluent samples
containing heavy metal degrading isolates, serial dilution was
done as Azad et al. (2013) to isolate desired bacteria. Streak
plate technique was followed during isolation. Control plates
also prepared with LB media without including any heavy
metal to make comparison. Colonies differing in morphologi-
cal characteristics were selected, picked, purified and then pre-
served on different plates for further studies.
Multiple metal resistance capacity
All isolates (S1, S2, S3, S4 and S5) were separately grown on
LB agar plates supplemented with Cd, Cr and Pb (300 lg/
mL) at pH 7.0 and 37 °C for 24 h; whereas after incubation
the resistance capacity of multiple heavy metals was assessed.
Relative effects of heavy metal consumption on microbial growth
The optimal growth conditions with reference to different
amounts of three heavy metals were determined. The isolates
were grown in a rotary shaker (Wise cubeÒ, Korea) at
150 rpm and pH 7.0, while the temperature was 37 °C in LB
broth medium supplemented with different types of heavy
Isolation and characterization of heavy-metal resistant bacteria from tannery effluent 67

Figure 1 Study area map indicating Chittagong City (A), Places of Madina Tannery (B,C) and three sampling sites (D).

Table 1 Details of effluent sampling location surroundings of (Shimadzu, Japan). After 6–8 h of incubation the effect of
Madina tannery. heavy metal concentration on their growth was assessed.
Sites Features of Sites Latitude Longitude
0 00
Determination of minimum inhibitory concentration (MIC)
Madina Tannery 22° 40 06.24 N 91° 810 88.7300 E
1 Nearby area of 3 Signal 22° 400 05.2400 N 91° 810 83.2600 E
Battalion Archery To asses MIC, heavy metal resistant selected isolates were
Range, Chittagong grown on heavy metal incorporated media against respective
2 Jalalabad Word no. 2, 22° 390 90.1700 N 91° 810 64.3800 E heavy metal; was identified by gently inclining the concentra-
Chittagong tion of the heavy metals (Pb, Cd and Cr) on LB agar plates
3 Nearby area of 22° 390 85.4100 N 91° 810 97.8500 E until the isolates failed to give colonies on the petri plate.
Chittagong-Rangamati- The starting concentration of the heavy metals was 50 lg/mL
Khagrachori Highway
and the culture growing on the final concentration was trans-
ferred to the higher concentration each time by streaking on
metals (gradually increasing 100 lg/mL at every time, until it the agar plate. When the isolates failed to grow on petri plate,
reaches to 1000 lg/mL) separately. The optical density (OD) MIC was assessed according to standard protocol of European
was measured (at k = 600 nm) using UV spectrophotometer food safety authority (EFSA), Parma, Italy, 2012.
68
Heavy metal biodegradability assay
Bacterial isolates were cultured into shake flask containing LB
broth medium for one hour in a rotary shaker at 150 rpm,
while pH and temperature were maintained 7.0 and 37 °C
respectively. After optical density is reached at 0.6
(k = 600 nm: for equal enzyme activity), then 100 ppm of ster-
ilized heavy metal (Pb, Cd or Cr) was added separately in every
culture flask and again incubated for 24 h at same condition.
Then total culture was centrifuged (Sigma 2-16KL, Germany)
at 5000 rpm for 15 min. The supernatants were separated and
mixed to the double volume of concentrated HNO3. Then
those mixtures were heated to 100 °C on a Hotplate Stirrer
(Lab tech-Daihan Company) to accomplish acid digestion
until the final volume decrease and down to initial supernatant
volume. Through a filter paper (Whatman 42) the extract was
filtered to remove any insoluble material and collected into a
volumetric flask and then diluted. This extract of total heavy
metal reduction was analyzed by Atomic Absorption Spec-
trophotometer (Shimadzu AA-7000, Japan) and the result
was compared with control to calculate heavy metal degrada-
tion capacity (%) as follows:
% of heavy metal utilized
Heavy metal utilized ðppmÞ
¼  100
Heavy metal added to the LB broth ðppmÞ
Heavy metal utilized ðppmÞ
¼ Heavy metal added to the LB broth ðppmÞ

Heavy metal at the end of culture ðppmÞ
Phenotypic and biochemical characterization of bacterial
isolates
The bacterial isolates were characterized based on cultural,
morphological and biochemical characteristics as described
in the Cowan and Steel’s Manual for the identification of Med-
ical Bacteria (Barrow and Feltham, 1993). For the activities of
oxidase, catalase, methyl red, indole production, citrate utiliza-
tion and carbohydrate (Glucose, Sucrose, Maltose, Xylose and
Lactose) utilization, isolates were biochemically analyzed
(Barrow and Feltham, 1993). According to Bergey’s Manual
of systemic Bacteriology the isolates were provisionally identi-
fied up to genus level (Claus and Berkeley, 1986).
Statistical analysis
Triplicate measurements were done in all the cases during the
observation and assessment of bacterial growth incorporated
with different levels of heavy metals. Data were captured into
Microsoft Excel Software, version 2010 which was used to cal-
culate means and standard deviations. Student’s t-test was
applied to confirm that the observed changes were statistically
significant.
Plasmid DNA extraction
Plasmid DNA extractions of heavy metal resistant bacteria
were done according to the alkaline lysis method (Sambrook
and Russel, 2001). Then visualized it on gel electrophoresis
L.W. Marzan et al.
at 1% agarose gel and compared with marker DNA (1kbp
sharp DNA Marker- RBC Bioscience) to assess plasmid DNA.
Results
Screening and isolation of heavy metal resistant bacteria
Visual observation of growth in heavy metal (Lead) supple-
mented (300 lg/mL) LB medium after 24 h of incubation indi-
cated that each of the collected sample consists of heavy metal
degrading bacteria, as they can grow there by degrading heavy
metals. After primary screening of the collected samples, serial
dilution was conducted to isolate desired bacteria (Table 2).
Total twenty bacterial isolates were isolated, among them five
isolates (S1, S2, S3, S4 and S5) were selected for further study.
Comparative analysis for multiple heavy metal degrading ability
To ascertain potential heavy metal degrading bacterial isolates,
we have conducted growth curve analysis for five isolates in LB
broth medium containing heavy metals (Pb, Cd or Cr, sepa-
rately), and their resistance capacity was assessed (data not
shown for S2 or S3). In this experiment, S1 and S4 showed
good tolerance capacity against multiple heavy metals.
Besides, S5 showed better tolerance to Pb and Cr, whereas it
showed sensitivity to Cd (Table 3).
Relative heavy metals consumption rate on bacterial growth
Hundred lg/mL amount of heavy metals (Cd, Cr or Pb, sepa-
rately) were supplemented in LB broth medium for each iso-
late, where each heavy metal concentration was gradually
increased from100 to 1000 lg/mL. The cultures were incubated
for 6–8 h and measured for optical density (at k = 600 nm) in
UV spectrophotometer to study the relative consumption rate
and bacterial resistance against each heavy metal. All bacteria
showed high tendency to decrease optical density while
increasing metal concentration (except Cr) in the medium
(Fig. 4; Table 3). Then three potential isolates (S1, S4 and
S5) have been selected for conducting further bioremediation
tests.
Assessment of MIC against each heavy metal
Minimum inhibitory concentration (MIC) for each heavy
metal was examined ranging from 50 to 1900 lg/mL. It was
found that all isolates exhibited resistance to heavy metals.
MIC of heavy metals showed highest tolerance to Pb, by the
selected isolates S1 and S4 (Table 3).
Table 2 Total viable cell count.
Dilution Heavy metal Control plate Percentage of
factor of (Pb) (without heavy heavy metal (Pb)
the sample incorporated metal-Pb) resistant bacteria
media
101 2.5  102 8.05  102 31.05%
102 0.8  103 5.03  103 35.78%
103 0.54  104 2.2  104 24.54%
Isolation and characterization of heavy-metal resistant bacteria from tannery effluent 69

and S4 showed 1.4 times higher (P < 0.01) than S1. On the
Table 3 Morphological and biochemical characteristics, car-
other hand, S1 showed 7.3 times (P < 0.01), as well as S4
bohydrate utilization test, heavy metal resistance capacity and
showed 5.5 times higher (P < 0.01) Cd degrading capacity
MIC of bacterial isolates (Barrow and Feltham, 1993; Claus
than S5 (Fig. 5).
and Berkeley, 1986).
Bacterial Isolates S1 S4 S5 Characterization and identification
Morphological characteristics
Colony color White Yellowish Whitish, not Top three potential heavy metal degrading isolates (S1, S4 and
Milky Brown milky S5) were characterized based on their cultural, morphological
Gram Nature Positive Positive Negative
and biochemical characteristics (Table 3). Compared with
Cell shape Round Round Rod
standard description of Bergey’s Manual of determinative bac-
Biochemical test results teriology 9th edition (Bergey et al., 1974; Williams and
Oxidase
+
Wilkins, 1994), the isolates were provisionally identified up
Catalase
+ + to genus level as Gemella sp. (S1); Micrococcus sp.(S4) and
Indole



Hafnia sp. (S5) are consistent with past field studies (Claus
Methyl-Red

+
Citrate +


and Berkeley, 1986).

Utilization of carbohydrate Plasmid DNA extraction

Glucose + +
Sucrose +
Maltose +
+ Only Gemella sp. (Fig. 6, Lane 1 and 2) and Micrococcus sp.
Xylose
(Fig. 6, Lane 3 and 4) harbor plasmid, while Hafnia sp. did
Lactose
not show any plasmid.
Resistance capacity
Pb +++ +++ ++ Discussion
+
Cr ++ ++ + Being an industrial city, Chittagong is facing pollution prob-
Cd +++ ++
lems. Heavy metals discharged from leather and other indus-
MIC (lg/ml
1) tries, as well as ship breaking yard, pose threat to human
Pb 1900 1800 1200 population, marine biodiversity as well as agricultural environ-
Cr 360 345 300 ment. Though, huge amounts of heavy metals in our environ-
Cd 1350 1100 50 ment cause a devastating effect; no effective remediation
Provisionally Identified Gemella Micrococcus Hafnia sp. technique has still been taken in hand. Bioremediation is
Bacteria sp. sp.
cost-effective, safe and eco-friendly; can be virtually restored
Claus and Berkeley
a solution to its pure condition (Liu et al., 1997). So, the major
(1986)
focus of this study is to isolate and identify the major bioreme-
diating bacterial agents that help the natural recovery of sur-
roundings (agricultural and residential environment) and
assess their comparative heavy metal remediation capacity to
select few isolates that can be a solution for recovering future
pollution by untreated heavy metal containing effluent.
Preliminary screening of the collected samples for heavy
metal resistance ability showed that all samples were positively
grown utilizing heavy metal (Pb) in their culture media. Serial
dilutions of all samples yield five (5) distinct isolates from the
heavy metal resistant bacterial population based on their mor-
phology (Table 2; Figs. 2 and 3). The bacterial isolates were
then characterized by morphological, biochemical tests, multi-
ple heavy metal resistance capacity, MIC and comparative
Figure 2 Culture plates (A) with and (B) without heavy metal
heavy metal degradation capacity. Identification of bacterial
incorporated media.
isolates were done (Table 3) according to Bergey’s Manual
of Determinative Bacteriology (Barrow and Feltham, 1993;
Bergey et al., 1974).
Assessment of heavy metal biodegradation capacity Depending on gram staining, two isolates (S1 and S5) were
identified as gram-positive and the other one (S4) as gram-
To measure total heavy metal (Pb, Cr or Cd) biodegradation negative bacteria (Table 3) by detecting peptidoglycan which
capacity, the treated samples were analyzed by Atomic is present in a thick layer in bacteria (Burke and Pister, 1986).
Absorption Spectrophotometer and compared with control. Micrococcus sp. are oxidase-positive, which can be used to
Among the isolates, S1 showed 3 times (P < 0.01) Lead distinguish them from other gram positive bacteria like most
(Pb) degrading ability and S4 showed 2 times higher Staphylococcus sp., which are generally oxidase-negative
(P < 0.01) compare to isolate S5. In the case of Cr, S1 showed (Thelwell et al., 1998). In our study, S4 (Micrococcus sp.)
1.7 times higher degrading ability (P < 0.05) compare to S5, was identified as oxidase positive as well as it may be
70
Figure 3 Pure cultures of five (S1, S2, S3, S4, S5) bacterial isolates.
Micrococcus luteus, since it produced yellow to brown colonies
(Table 3) on growth medium compared to the red colony of
Micrococcus roseus. Gram staining, oxidase tests and carbohy-
drate utilization studies showed (Table 3) similarities of S1
with Gemella sp., which is gram positive, oxidase negative
and utilizes all carbohydrates (Nucifora et al., 1989).
A coliform is an aerobic or facultative anaerobic rod and
gram negative, which as identified by IMViC test can produces
gas from lactose within 48 h. These coliforms indicate fecal
contamination in the previous study, where it has been done
to confirm the presence of coliform bacteria in industrial efflu-
ent samples (Malik and Jaiswal, 2010). Our study found S5 is
coliform bacteria and identified as Hafnia sp. (data not
shown). More specific study with some other industrial water
effluents, established the presence of Hafnia alvei in tannery
effluent, which is very much deleterious to our environment
(Stackebrandt et al., 1982).
The multi-metal resistance capacity approached two bacte-
rial isolates Gemella sp. and Micrococcus sp. are highly resis-
tant to Pb and Cd compared to Hafnia sp. (Table 3).
Besides, Cd is a more lethal heavy metal for Hafnia sp.
(Table 3). Upon above experiments, the resistance level
Pb > Cd > Cr showed for Gemella sp. and Micrococcus sp.
It was also reported that the tolerant levels of heavy metal
for sewage bacteria Pseudomonas aeruginosa, Acinetobacter
resistance, Proteus vulgaris were shown to be Pb > Cd > Cr
(Powers and Latt, 1977).
Relative effects of bacterial growth in presence of heavy
metal(s) in different concentrations (100–1000 lg/mL) were
studied and it was observed that bacterial growth is concentra-
tion dependent, since it showed decreasing optical density (at
k = 600 nm) in accordance with the increasing heavy metal
concentration (Fig. 4). Besides, Hafnia sp. shows sensitivity
to Cd as well as their resistance capacity against Pb and Cr
are also lower compared to other bacteria. Interestingly,
Gemalla sp. and Micrococcus sp. show resistance and tolerance
capacity against Pb and Cr. On the other hand the result of Cd
resistance is insignificant (Fig. 4C).
Minimum inhibitory concentration (MIC) is the lowest
concentration at which the isolate is completely suppressed
L.W. Marzan et al.
(as demonstrated by the absence of visible bacterial growth)
is recorded. In this study order of MICs for the isolates S1
and S4 was found to be Pb > Cd > Cr and Pb > Cr > Cd
for the isolate S5 (Table 3). Gemella sp. is resistant against
Cd with MIC of 128 lg/mL, Cr with MIC of 1024 lg/mL
has been recently shown by Ashour et al. (2011). Gemella sp.
was characterized in our study with MIC against Cd
1350 lg/mL, Cr 360 lg/mL as well as Pb 1900 lg/mL (Table 3).
Janda (2006) demonstrated that 13 bacteria are resistant to
heavy metals (Zn, Pb, Cr, Cd); where Micrococcus luteus was
found to be the most multiple heavy metals resistant. Our
study found the multiple heavy metal tolerance capacity for
Micrococcus sp.; with MIC against Pb (1800 lg/mL), Cr
(345 lg/mL) and Cd (1100 lg/mL) being the second highest
capacity (Table 3). Among three characterized bacteria, Hafnia
sp. was identified here as the lowest capacity of Pb and Cr
reducing bacteria, was also sensitive to Cd and is similar to
the recent studies by Fakhruddin et al. (2009) and Resende
and Silva (2012).
The isolates measured by Atomic Absorption Spec-
trophometer; Gemella sp. and Micrococcus sp. showed consid-
erable degradation of Pb which were 55.16 ± 0.06% and
36.55 ± 0.01%, respectively. On the other hand Hafnia sp.
shows very low degradation capacity (18.28 ± 0.06%). Degra-
dation of Cd by Gemella sp. and Micrococcus sp. showed
50.99 ± 0.01% and 38.64 ± 0.06% respectively, where Hafnia
sp. was Cd sensitive. But degradation of Cr was moderate for
all isolates which showed 6.14 ± 0.24, 8.42 ± 0.02 and 3.69
± 0.2% for Gemella sp., Micrococcus sp. and Hafnia sp.
respectively. It might be due to existence here of hexavalent
Chromium (Cr6+), which is known to be 100–1000 times more
toxic than trivalent (Cr3+) form (Gauglhofer and Bianchi,
1991). So, it is found here, conversion of Cr6+ may not be
much easier with these three bacteria, where waste water
detoxification mechanisms by microorganism are an important
factor.
Bacterial plasmids encode resistance systems for toxic metal
ions are inherited by plasmids in many bacteria (Silver and
Phung, 1996). Recent study shows heavy metal resistance
capacity either plasmid mediated or chromosomal DNA
Isolation and characterization of heavy-metal resistant bacteria from tannery effluent 71

Figure 4 Optical density (k = 600 nm) was measured at UV Spectrophotometer (Shimadzu, Japan) after 6–8 h incubation in LB broth
medium incorporated with heavy metals as Pb (A), Cr (B) and Cd (C) to observe relative heavy metal consumption rate on the growth of
bacterial isolates.

mediated (Virender et al., 2010). For determination of genetic
basis for metal resistance, plasmid profiling is important. Plas-
mid DNA extraction of three bacterial isolates having
biodegradation capacity was assessed to understand whether
their heavy-metal resistance capacity is plasmid DNA or chro-
mosomal DNA mediated. In our study, two strains Gemella sp.
and Micrococcus sp. showed plasmid DNA, while Hafnia sp.
does not harbor plasmid (Fig. 6). Probably the high degrading
capacity of Gamella sp. and Micrococcus sp. can be the reason
for their plasmid retaining ability (Ghosh et al., 1997), where
Hafnia sp. has lower degrading ability without plasmid
DNA. In bacteria, the heavy metal resistant genes are located
either on the bacterial chromosome or in the plasmids or on
both (Nies and Brown, 1997). According to Malik (2004),
Cd and Cr resistant genes are present in plasmid DNA but
Pb resistance gene is located on chromosomal DNA of Enter-
obacteria. In this way, chromosomal gene might be responsible
for this kind of lower degrading capability but more usually
conferring resistance are located on plasmid (Woertz and
Mergeny, 1997). Although this fundamental study will support
for plasmid curing, transformation and evaluation of heavy
metal resistance can pave way for the genetic basis of heavy
metal resistant mechanism. Plasmid mediated heavy metal
resistance is important for further transformation study, which
will render any heavy metal sensitive bacteria (recipient) into
being heavy metal resistant bacteria (Mergeay et al., 2003;
Vaijiheh and Naser, 2003).
Further study of the effects of different supplements and
conditions in their growth is needed to identify their efficiency
as bioremediation agents, where optimization of pH, tempera-
ture, and incubation time can influence metal resistance capac-
ity (Shivakumar et al., 2014).
To make it usable for local farmers in their paddy fields and
hatcheries, this is the base study to develop three biological
72 L.W. Marzan et al.

Figure 5 Heavy metals’ (Pb, Cr or Cd) degradation capacity (%) by each bacterial isolates. Triplicate measurements were done and
compared with control in each case. Error bars indicate ±SD. Total heavy metal reduction was analyzed by Atomic Absorption
Spectrophotometer.

M 1 2 3 4 5 6

10000bp
8000bp
7000bp
6000bp
5000bp
4000bp
3000bp
2800 bp (approx. )
2000bp 1900 bp (approx. )

1000bp

500bp

Figure 6 Lane 1 and 2 show plasmid of Gemella sp.; 3 and 4 show plasmid of Micrococcus sp.; 5 and 6 shows no plasmid band of
Hafnia sp.

agents with resistance and degradation capacity, focusing tan-
nery effluent pollution, might be helpful to formulate and
develop local production of bioremediation agents for human,
agricultural and aquatic environmental cleaning.
Conclusion
presented in this study support the concept that three bacteria
(Gemella sp. Micrococcus sp. and Hafnia sp.) had significant
bioremediation potentiality which might be used to formulate
bioremediation agents to detoxify tannery effluents at
industrial surroundings in the natural environments in
Bangladesh.

In this study, twenty samples were collected from tannery
industrial surroundings in three contaminated sites and their
heavy metal degrading potentiality was assessed. From those
samples, five (5) bacterial isolates have been selected. The iso-
lates were subjected to study multiple metal resistance capac-
ity, growth curve analysis on different concentrations of
heavy metals, MIC and heavy metal biodegradability analysis
to select the best candidates that might be further used for
bioremediation of heavy metal pollutants. Depending on vari-
ous tests for biochemical characterization; we identified them
as Gemella sp. Micrococcus sp. and Hafnia sp. All the results
Conflict of interest
No conflict of interest influenced in this research.
Authors’ contribution
LWM conceived and designed the project and MH carried out
the laboratory experiments. LWM, SAM and YAR prepared
the manuscript. LWM supervised and interpreted the results.
All authors read and approved the final manuscript.
Isolation and characterization of heavy-metal resistant bacteria from tannery effluent
Acknowledgement
The authors are grateful to Research and Publication Office,
University of Chittagong, Chittagong-4331, Bangladesh for
partial financial assistance (Memo No. 5628/2015; fiscal
year 2014–2015). The authors are pleased to mention about
the fruitful suggestions for the grammar check from Mahira
Taj.
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