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Dhaka Univ. J. Biol. Sci. 26(1): 29-38, 2017 ( January)
CADMIUM AND LEAD TOLERANT BACTERIA ISOLATED FROM
INDUSTRIAL WASTE WATER
MD ARIFUR RAHMAN BHUIYAN, TAZEEN FATIMA KHAN,
SHAHJAHAN CHOUDHURY AND SM IMAMUL HUQ*
Bangladesh-Australia Centre for Environmental Research, Department of Soil, Water and
Environment, University of Dhaka, Dhaka-1000, Bangladesh
Key words: Heavy metal, Waste water, Bacteria, Metal resistant
Abstract
An in vitro study was conducted to isolate, identify and characterize heavy
metal resistant bacteria from industrial waste water and to determine their
tolerance capacity to cadmium and lead. Different morphological, physiological
and biochemical tests were carried out to identify the bacterial isolates in the
waste water. A total of eight bacterial isolates viz., Staphyllococcus intermedius,
Pseudomonas aeruginosa, Bacillus cereus, Bacillus subtilis, Escherichia coli,
Acinetobacter baumanii, Pseudomonas flavescens and Acinetobacter lwofii were
identified from metal polluted tannery and steel industrial areas. Bacterial
response to cadmium tolerance was determined by treating them with CdCl2
solution at a rate of 1, 2, 2.5 and 5 µg/ml. In case of lead tolerant bacteria, PbCl2
solution was applied at a rate of 0.05, 0.125, 0.2 and 0.5 µg/ml. Pseudomonas
aeruginosa isolated from waste water of steel industries and Bacillus cereus from
tannery area was found to be the most tolerant species to the different doses of
cadmium and lead. The study indicated that Pseudomonas aeruginosa and Bacillus
cereus could be good candidates for the treatment and elimination of heavy
metals from industrial waste water. The present study may be helpful to the
bioremediation of heavy metals in the contaminated environment.
Introduction
Heavy metal contamination due to natural and anthropogenic sources is a global
environmental concern. Human activities, such as mining operations and the discharge
of industrial wastes, result in the accumulation of metals in the environment and are
eventually accumulated through the food chain, leading to serious ecological and health
problems(1-2). The pollution of the ecosystem by heavy metals is a real threat to the
environment because metals cannot be naturally degraded like organic pollutants and
persist in the ecosystem being accumulated in different parts of the food chain(3). These
heavy metals influence the microbial population by affecting their growth, morphology,
biochemical activities and ultimately resulting in decreased biomass and diversity(4).
*Author for correspondence: <imamhuq@hotmail.com>.
30 BHUIYAN et al.
Toxic metal tolerance of bacteria has been studied for many years but considering the
range of toxic metal ions and diversity of microbes, the overall efforts appear to be
limited. However, there is no general mechanism for tolerance to all toxic metals(5), as
there are evidences for both chromosomal and plasmid borne genes for tolerance(6). The
ability of microbial strains to grow in the presence of heavy metals would be helpful in
the wastewater treatment where microorganisms are directly involved in the
decomposition of organic matter in biological processes for wastewater treatment,
because often the inhibitory effect of heavy metals is a common phenomenon that occurs
in the biological treatment of wastewater and sewage(7).
Most of the studies regarding the tolerance of heavy metal toxicity by micro-
organisms were focused on soil quality. Therefore, the effects of heavy metals on bacteria
living in wastewater need to be studied. The current study aimed at assessing the
tolerance capacity of bacterial isolates present in industrial wastewater to the heavy
metals particularly cadmium and lead.
Materials and Methods
Sampling sites: The steel industrial area in Savar and tannery area in Hazaribagh, both
places being adjacent to Dhaka, were selected for the collection of waste water samples
that were contaminated with steel industry effluents, waste water and tannery effluents.
Waste water was collected from drains as well as from corresponding rivers in which
waste water was discharged. The georeferences of the sampling sites in tannery area are
23.738961ºN and 90.367615º E (Hazaribagh Tannery intersection); 23.734852º N and
90.354983º E (Buriganga River). The georeferences of the sampling sites in steel industrial
area are 23.846188º N and 90.243359◦ E (Ulail, Savar bazaar); 23.845975º N and 90.242471º
E (Dhalshwari River).
Collection and preservation of waste water samples: Sterilized sampling bottles were
used to collect waste water. The bottles were rinsed 2 to 3 times with the water being
collected, and then these were filled up to the top and sealed carefully so that no air
could enter into the bottles. Collection of water samples were done by following
composite sampling techniques as described in Imamul Huq and Alam(8). For
microbiological analysis, collected samples were kept in cool and dark place before and
after the analysis so the sample does not deteriorate and the analytical results were
representative.
Determination of cadmium and lead concentrations in waste water samples: To determine
the total cadmium and lead concentration present in the water samples, the water
samples were digested using "Aqua Regia" (HNO3 : HCl = 1 : 3) solution. Waste water
sample was mixed with aqua regia solution at a ratio of 10 : 1 followed by heating in a
digestion chamber. When the solution became one third of its initial volume, the sample
was thoroughly treated with distilled water, filtered and collected into 50 ml volumetric
CADMIUM AND LEAD TOLERANT BACTERIA 31
flask. The concentration of total cadmium and lead was determined by using a VARIAN
Atomic Absorption Spectrophotometer (AAS)(8). For every ten samples a Certified
Reference Material (CRM) was included to ensure quality control/quality assurance
(QC/QA).
Culture of bacteria: For culture of bacteria, simple "Pour Plate Technique" was
followed as described by Khan and Huq(9).
Isolation of cadmium and lead tolerant bacteria: For isolation of cadmium and lead
tolerant bacteria, unsterilized waste water samples were used as source to grow the
bacteria. First, bacteria were grown on solidified agar plates and were kept in incubation
at 37ºC for 24 hrs. Then purification of the isolates (pure culture) was done. At the same
time a portion of the collected waste water samples was sterilized to kill the existing
bacteria but concentration of heavy metals remained same. This sterilized water was then
applied on bacteria to test their metal tolerance. For this purpose, 5 ml of the sterilized
waste water was applied to each of the isolates and then incubated for 3 days at 37ºC. The
isolates that were capable of growing new colonies within this time span were
considered as heavy metal tolerant, particularly cadmium and lead tolerant(10). Following
the colony morphology, gram staining, spore staining and relevant biochemical tests,
isolated bacteria were identified according to the "Bergey’s Manual of Determinative
Bacteriology"(11).
Determination of bacterial response to cadmium and lead tolerance: Cadmium and lead
containing salts, namely CdCl2.H2O and PbCl2 were used to prepare 1000 mg/l stock
solution of each. Each stock solution was filter-sterilized and added to the nutrient broth
media at varying concentrations of metals to determine their tolerance capacity(12). The
concentrations of heavy metals applied to assess the tolerance of the isolated bacteria
were determined on the basis of the background concentration of waste water. For this,
concentrations of 1, 2, 2.5 and 5 µg/ml of CdCl2 and 0.05, 0.125, 0.2 and 0.5 µg/ml of PbCl2
were added in media. These concentrations were actually 50% higher than sample
concentrations. In case of control, no treatments were used. Then identified isolates were
incubated in duplicate tubes containing heavy metals of different concentrations added
with nutrient broth and incubated at 370C for 24, 48, 72, 96 and 120 hrs, respectively.
After incubation, growth of bacteria in liquid media was confirmed by the formation of
white precipitate. At 24 hrs intervals, the optical density of each of the samples were
determined at 580 nm by using spectrophotometer in order to determine the growth
response of each isolates treated with different concentration of cadmium and lead(13).
Statistical analysis: The experimental data were statistically analyzed by using the
Microsoft Excel and STATA (version 12). T-test was done to know whether or not there
was significant difference among the selected bacteria.
32 BHUIYAN et al.
Results and Discussion
Bacterial response to cadmium tolerance: A total of four bacterial species were identified
as cadmium and lead tolerant from the waste water samples: these were Staphyllococcus
intermedius, Pseudomonas aeruginosa, Bacillus cereus and Acinetobacter baumanii. The results
of the optical density of the four bacterial species each treated with 1, 2, 2.5 and 5 µg/ml
of CdCl2 for the period of 24, 48, 72, 96 and 120 hrs are shown in Figs 1-4.
Fig. 1.
Growth of
Staphyllococcus intermedius
at different concentrationsof CdCl2.
Fig. 2.
Growth of
Pseudomonas aeruginosa
at
different concentrations of CdCl2.
Fig. 3.
Growth of
Bacillus cereus
at different
concentrations of CdCl2.
Fig. 4.
Growth of
Acinetobacter baumanii
at
different concentrations of CdCl2.
It is clear from the Fig. 1 that initially the growth continued for 120 hrs (5 days) at 1
µg/ml concentration but the growth decreased at 5 µg/ml. Bruins et al. (14) reported that,
Staphyllococcus aureus strains treated with Cd2+ showed highest resistance probably
CADMIUM AND LEAD TOLERANT BACTERIA 33
caused by the efflux pumps. Figure 2 indicates that the growth response of Pseudomonas
aeruginosa at different concentrations of CdCl2 was satisfactory and it showed its best
response at 5 µg/ml at which the growth seemed to be increasing even after 120 hrs. The
growth responses at other concentrations were also satisfactory and it showed its
moderate response at 1 and 2 µg/ml.
Hassen et al.(15) and Hussein et al.(16) isolated a Cd-resistant Pseudomonas aeruginosa
with minimum inhibitory concentration of 1.5 mM in nutrient broth. On the other hand,
Malik(17) reported that Pseudomonas strain - H1 isolated from soil was resistant to 225 ppm
Cd2+. The growth response of Bacillus cereus (Fig. 3) continued to increase upto 2.5 µg/ml
but after that it dropped at the highest concentration of 5 µg/ml. The growth nearly
leveled off after 96 hrs at 5 µg/ml concentration but the overall growth response at
different concentrations of CdCl2 was moderately satisfactory. Although the metal
tolerance response by Acinetobacter baumanii was initially satisfactory (Fig. 4) upto 2
µg/ml but after that the curves tended to decrease. However, the growth rapidly
decreased at 5 µg/ml and seemed to be almost leveled after 96 hrs (4 days).
Raja(18) reported that Acinetobacter radioresistens isolated from sewage water collected
in and around Madurai district, South India was resistant to Cd (4 - 7 mM), Cr (0.7 mM),
Ni (6.75 - 8.5 mM), Pb (6 mM), As (6.5 - 15 mM) and Hg (0.75 mM).
Bacterial response to lead tolerance: Four metal tolerant bacteria were treated at a rate of
0.05, 0.125, 0.2 and 0.5 µg/ml of PbCl2 solution and incubated for 24, 48, 72, 96 and 120
hrs. Then optical density was measured at 24 hrs intervals which indicated the metal
tolerance response of the isolates. The results of the optical density are shown in Figs 5 - 8.
It is evident from Fig. 5 that initially the tolerance response of Staphyllococcus
intermedius was satisfactory at 0.05 and 0.125 µg/ml of PbCl2 but after that the growth
response was drastically reduced at 0.2 and 0.5 µg/ml. It is observed from Fig. 6 that
Pseudomonas aeruginosa responded best at 0.5 µg/ml PbCl2 concentration and the growth
seemed to be continuing even after 120 hrs (5 days). Selvi et al. (19) reported that more than
80% of Pseudomonas sp., 70% of the E. coli, 50% of Bacillus, 90% of Flavobacterium and 90%
of Alcaligens isolated from tannery effluents were found resistant against lead. Minimum
inhibitory concentrations (MIC) of lead was found to be 60 - 80, 80 and 70 ppm for
Escherichia coli, Alcaligenes sp. and Pseudomonas sp., respectively.
It is clear from Fig. 7 that the growth response of Bacillus cereus at different
concentrations indicated a higher metal tolerance response. It showed its best growth
response at concentration of 0.5 µg/ml PbCl2 and the growth seemed to be continuing
even after 120 hours (5 days). Growth responses at other concentrations were also
satisfactory. According to Murthy et al.(20) Bacillus cereus showed remarkable ability to
remove lead from media. Lead concentrations of 200, 300, 400 and 500 mg/l were reduced
to 75.6, 56.7, 41.8 and 39.1%, respectively, from the medium after 72 hrs. It is clear from
34 BHUIYAN et al.
Fig. 8 that Acinetobacter baumanii showed its best growth response at 0.5 µg/ml. However,
the growth response was more or less moderate at other concentrations.
Fig. 5. Growth of
Staphyllococcus intermedius
at different concentrations of CdCl2.
Fig. 6. Growth of
Pseudomonas aeruginosa
at
different concentrations of CdCl2.
Fig. 7. Growth of
Bacillus cereus
at different
concentrations of CdCl2.
Fig. 8. Growth of
Acinetobacter
baumanii
at
different concentrations of CdCl2.
Comparative cadmium and lead tolerance of the isolated bacteria: Optical density of the
four bacterial isolates was measured at the highest concentration levels (5 µg/ml for
CdCl2 and 0.5 µg/ml for PbCl2). Comparison of cadmium and lead tolerance capacity of
these isolates are shown in Figs 9-10.
It is evident from Fig. 9 that Pseudomonas aeruginosa showed its highest tolerance
response to CdCl2 at 5 µg/ml and the growth seemed to be continuing even after 120 hrs
(5 days). The least tolerance to CdCl2 was shown by Acinetobacter baumanii. On the
contrary, Bacillus cereus showed an initial growth response up to 72 hrs but after that the
growth leveled off. Staphyllococcus intermedius showed moderate response for cadmium
and the trend seemed to be almost similar with that of Bacillus cereus. Both Bacillus cereus
CADMIUM AND LEAD TOLERANT BACTERIA 35
and Staphyllococcus intermedius showed moderate tolerance to 5 µg/ml of CdCl2. Bacteria
of the genus Pseudomonas are well-studied and are of great interest not only because of
their high resistance to heavy metals and other toxic substances, but also for their simple
nutritional requirements and rapid growth on standard laboratory media(21). In the
laboratory, Pseudomonas aeruginosa showed resistance to high concentrations of Zn, Cu,
Ni, Pb, Cd and Hg. Whatever may be the case, the highest cadmium tolerance response
was observed in the bacteria - Pseudomonas aeruginosa(22-23).
Fig. 9. Comparison of growth response of the isolates at 5 µg/ml CdCl2.
Fig. 10. Comparison of growth response of the isolates at 0.5 µg/ml PbCl2.
Figure 10 clearly shows that among the four isolates, Bacillus cereus showed its best
performance and the highest tolerance response at 0.5 µg/ml of PbCl2 solution. The
reasons could be that high metal concentration influences increased enzyme activity,
increased respiration rate as well as normal regeneration system. The second best
tolerance response was observed in Pseudomonas aeruginosa and the trend seemed to be
Staphyllococcus
intermedius
Pseudomonas
aeruginosa
Bacillus cereus
Acinetobacter
baumanii
Staphyllococcus
intermedius
Pseudomonas
aeruginosa
Bacillus cereus
Acinetobacter
baumanii
36 BHUIYAN et al.
continuing after 120 hrs (5 days) and it did not show any declining trend even after that
time. Acinetobacter baumanii showed its moderate tolerance response against 0.5 µg/ml of
PbCl2. The least tolerance response was observed for Staphyllococcus intermedius. Its
highest tolerance response was observed at 48 hrs, but after that the trend seemed to be
almost leveled off indicating its lowest tolerance capacity against lead. According to
Murthy et al.(20) the lead biosorption capability of Bacillus cereus was studied at different
concentrations (100, 200, 300, 400 and 500 mg/l) of lead. Studies revealed that an
optimum temperature of 30°C and pH of 5.0 facilitate the maximum biosorption of lead
by 24 hrs old culture of Bacillus cereus.
The removal of lead by Bacillus cereus revealed that the percentage of metal removed
by the bacteria decreases with increasing concentration of lead. The enhancement in
metal sorption could be due to the increase in electrostatic interactions involving sites of
progressively lower affinity for metal ions. Toxicity testing in liquid media allows a good
evaluation of metal toxicity in polluted environments, such as industrial effluents and
sewage sludge leachates(24). Liquid media toxicity testing is different from toxicity testing
on solid media, where the conditions of diffusion, complexation and availability of
metals are different from those in solid media.
Hassen et al.(24) also tested the levels of tolerance of environmental bacteria to the
different divalent metal ions including Cu2+, Co2+, Cd2+ and Zn2+ in nutrient broth. He
reported that the test in liquid media was sensitive at concentrations that are 10 to 1000
times lower than those obtained in solid media. Scientists revealed that most bacterial
isolates were resistant to very high concentrations of heavy metals regardless of the level
of metal concentrations in their environment. They also discovered that the most
predominant isolates at high concentrations of the metal ions include Bacillus spp.,
Pseudomonas spp., Corynebacterium spp., Micrococcus spp., Flavobacterium spp., Proteus,
Citrobacter, Alcaligenes and Enterobacter(25).
In case of cadmium tolerance, the t-test indicated that Staphyllococcus intermedius
showed significant difference (p = 0.0058, t = 4.9305 and p = 0.001, t = 7.4726) with rest of
the bacteria whereas it showed no significant difference with Bacillus cereus (p = 0.8973,
t = 0.1352). In case of lead tolerance, the t-test showed significant differences (p = 0.003,
t = 6.2073; p = 0.0122, t = 3.2232 and p = 0.0352, t = 2.5313) among Bacillus cereus,
Pseudomonas aeruginosa and Acinetobacter baumanii.
Although the conditions in which bacteria grow actually differ from laboratory to
natural environment yet it can be concluded from the study that both Pseudomonas
aeruginosa and Bacillus cereus could be used in bioremediation of metal contaminated
waste water particularly cadmium (Cd) and lead (Pb).
The presence of these metals in the environment can alter the biomass, diversity,
population as well as growth and survival of bacterial communities. The present study
CADMIUM AND LEAD TOLERANT BACTERIA 37
might provide a basis for broader investigation of metal tolerant bacteria considering
their potential use towards bioremediation.
Acknowledgements
The first author is grateful to the Ministry of Science and Technology, Government of
the People’s Republic of Bangladesh, for providing an S & T scholarship to financially
support the present research.
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(Manuscript received on 2 October, 2016; revised on 12 December, 2016)
