Masudul Azad Chowdhury Department of Genetic Engineering and Biotechnology, Faculty of Biological Sciences, University of Chittagong, Chittagong 4331, Bangladesh Received 27 June 2016; r
Trang 1Isolation and biochemical characterization of
heavy-metal resistant bacteria from tannery effluent
in Chittagong city, Bangladesh: Bioremediation
viewpoint
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
KEYWORDS
Gemella sp.;
Micrococcus sp.;
Hafnia sp.;
Heavy metal resistance;
Degradation capacity;
Bioremediation;
Characterization
Abstract Toxic, mutagenic and carcinogenic heavy metals from tannery industries cause the pol-lution of agricultural environment and natural water sources This study aims to isolate, investigate and identify naturally occurring bacteria capable of reducing and detoxifying heavy metals (Chro-mium, Cadmium and Lead) from tannery effluent Three isolates were identified up to genus level based on their morphological, cultural, physiological and biochemical characteristics as Gemella sp., Micrococcus sp and Hafnia sp Among them Gemella sp and Micrococcus sp showed resis-tance to Lead (Pb), chromium (Cr) and cadmium (Cd), where Hafnia sp showed sensitivity to cad-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 Micrococcussp (38.64 ± 0.06%) Heavy metals degradation capacity of Gemella sp and Micrococ-cussp 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
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Introduction
Air, water and land which are the essential elements of life are contaminated constantly due to increasing population, rapid urbanization and industrialization (Chhikara and Dhankhar,
* Corresponding author Fax: +880 31 2606145, +880 31 2606014.
E-mail addresses: marzan.geb@cu.ac.bd , lmarzancu@yahoo.com
(L.W Marzan).
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and Fisheries.
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Egyptian Journal of Aquatic Research
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http://dx.doi.org/10.1016/j.ejar.2016.11.002
Trang 22008) 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 agricultunatu-ral 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)
(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 respiradetoxifica-tion mechanism using heavy metals and thus
become resistant to it (Ezaka and Anyanwa, 2011) The
isola-tion and characterizaisola-tion 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
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,
300lg/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: pH7.00) 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 (300lg/ 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
Trang 3metals (gradually increasing 100lg/mL at every time, until it
reaches to 1000lg/mL) separately The optical density (OD)
was measured (at k = 600 nm) using UV spectrophotometer
(Shimadzu, Japan) After 6–8 h of incubation the effect of heavy metal concentration on their growth was assessed Determination of minimum inhibitory concentration (MIC)
To asses MIC, heavy metal resistant selected isolates were grown on heavy metal incorporated media against respective heavy metal; was identified by gently inclining the concentra-tion of the heavy metals (Pb, Cd and Cr) on LB agar plates until the isolates failed to give colonies on the petri plate The starting concentration of the heavy metals was 50lg/mL and the culture growing on the final concentration was trans-ferred to the higher concentration each time by streaking on the agar plate When the isolates failed to grow on petri plate, MIC was assessed according to standard protocol ofEuropean food safety authority (EFSA), Parma, Italy, 2012
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
Madina tannery
Sites Features of Sites Latitude Longitude
Madina Tannery 22 ° 40 006.2400N 91° 81 088.7300E
1 Nearby area of 3 Signal
Battalion Archery
Range, Chittagong
22 ° 40 005.2400N 91° 81 083.2600E
2 Jalalabad Word no 2,
Chittagong
22 ° 39 090.1700N 91° 81 064.3800E
3 Nearby area of
Chittagong-Rangamati-Khagrachori Highway
22 ° 39 085.4100N 91° 81 097.8500E
Trang 4Heavy 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 added to the LB brothðppmÞ 100
Heavy metal utilizedðppmÞ
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
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 (300lg/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
Hundredlg/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 1000lg/mL The cultures were incubated for 6–8 h and measured for optical density (atk = 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 1900lg/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 factor of the sample
Heavy metal (Pb) incorporated media
Control plate (without heavy metal-Pb)
Percentage of heavy metal (Pb) resistant bacteria
10 1 2.5 10 2 8.05 10 2 31.05%
10 2 0.8 10 3 5.03 10 3 35.78%
10 3 0.54 10 4 2.2 10 4 24.54%
Trang 5Assessment of heavy metal biodegradation capacity
To measure total heavy metal (Pb, Cr or Cd) biodegradation
capacity, the treated samples were analyzed by Atomic
Absorption Spectrophotometer and compared with control
Among the isolates, S1 showed 3 times (P < 0.01) Lead
(Pb) degrading ability and S4 showed 2 times higher
(P < 0.01) compare to isolate S5 In the case of Cr, S1 showed
1.7 times higher degrading ability (P < 0.05) compare to S5,
and S4 showed1.4 times higher (P < 0.01) than S1 On the other hand, S1 showed 7.3 times (P < 0.01), as well as S4 showed5.5 times higher (P < 0.01) Cd degrading capacity than S5 (Fig 5)
Characterization and identification Top three potential heavy metal degrading isolates (S1, S4 and S5) were characterized based on their cultural, morphological and biochemical characteristics (Table 3) Compared with standard description of Bergey’s Manual of determinative bac-teriology 9th edition (Bergey et al., 1974; Williams and Wilkins, 1994), the isolates were provisionally identified up
to genus level as Gemella sp (S1); Micrococcus sp.(S4) and Hafnia sp (S5) are consistent with past field studies (Claus and Berkeley, 1986)
Plasmid DNA extraction Only Gemella sp (Fig 6, Lane 1 and 2) and Micrococcus sp (Fig 6, Lane 3 and 4) harbor plasmid, while Hafnia sp did not show any plasmid
Discussion Being an industrial city, Chittagong is facing pollution prob-lems Heavy metals discharged from leather and other indus-tries, as well as ship breaking yard, pose threat to human population, marine biodiversity as well as agricultural ment Though, huge amounts of heavy metals in our environ-ment cause a devastating effect; no effective remediation technique has still been taken in hand Bioremediation is cost-effective, safe and eco-friendly; can be virtually restored
a solution to its pure condition (Liu et al., 1997) So, the major 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 heavy metal degradation capacity Identification of bacterial isolates were done (Table 3) according to Bergey’s Manual
of Determinative Bacteriology (Barrow and Feltham, 1993; Bergey et al., 1974)
Depending on gram staining, two isolates (S1 and S5) were identified as positive and the other one (S4) as gram-negative bacteria (Table 3) by detecting peptidoglycan which
is present in a thick layer in bacteria (Burke and Pister, 1986) Micrococcussp are oxidase-positive, which can be used to distinguish them from other gram positive bacteria like most Staphylococcus sp., which are generally oxidase-negative (Thelwell et al., 1998) In our study, S4 (Micrococcus sp.) was identified as oxidase positive as well as it may be
Table 3 Morphological and biochemical characteristics,
car-bohydrate utilization test, heavy metal resistance capacity and
MIC of bacterial isolates (Barrow and Feltham, 1993; Claus
and Berkeley, 1986)
Morphological characteristics
Milky
Yellowish Brown
Whitish, not milky Gram Nature Positive Positive Negative
Biochemical test results
Utilization of carbohydrate
Resistance capacity
+
MIC ( lg/ml 1 )
Provisionally Identified
Bacteria
Gemella sp.
Micrococcus sp.
Hafnia sp.
Claus and Berkeley
(1986)
Figure 2 Culture plates (A) with and (B) without heavy metal
incorporated media
Trang 6Micrococcus 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 vulgariswere shown to be Pb > Cd > Cr
(Powers and Latt, 1977)
Relative effects of bacterial growth in presence of heavy
metal(s) in different concentrations (100–1000lg/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,
Gemallasp 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
(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
has been recently shown byAshour et al (2011) Gemella sp was characterized in our study with MIC against Cd
1350lg/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 (1800lg/mL), Cr (345lg/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
Figure 3 Pure cultures of five (S1, S2, S3, S4, S5) bacterial isolates
Trang 7mediated (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
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
Trang 8agents 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
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
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
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
1900 bp (approx )
2800 bp (approx )
500bp 1000bp 2000bp 3000bp 4000bp5000bp 6000bp 7000bp8000bp 10000bp
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 Hafniasp
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
Trang 9The 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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