Isolation and characterization of zinc resistant bacteria from a coil coating wastetreatment plant

INTERNATIONAL JOURNAL OF ENVIRONMENTAL SCIENCES Volume 5, No 5, 2015
© Copyright by the authors - Licensee IPA- Under Creative Commons license 3.0
Research article ISSN 0976 – 4402
Received on December2014 Published on March 2015 1030
Isolation and characterization of zinc resistant bacteria from a coil coating
industrial wastewater treatment plant
Joshua B. Owolabi and Melanie M. Hekeu
Department of Biological Sciences, Microbiology Unit, School of Natural and Applied
Sciences, College of Science and Technology, Covenant University,
P.M.B. 1023 Ota, Ogun State, Nigeria.
joshua.owolabi@covenantuniversity.edu.ng
doi: 10.6088/ijes.2014050100097
ABSTRACT
Wastewater effluents from metal coil coating industry containing priority pollutants including
chromium, cyanide, zinc, aluminum, cadmium, lead, nickel, selenium, tin, and mercury; oil
and grease, or total suspended solids are often discharged with limited or no treatment into
the environment thereby threatening microbial diversity and health of plants, animals and
humans. This study was carried out to assess the incidence of Zn2+
resistant bacteria from a
coil coating industrial wastewater treatment plant in Ota, Southwest Nigeria. In this study, ten
Zn2+
resistant bacterial strains were recovered from treated wastewater samples on nutrient
agar supplemented with 1mM concentration of zinc sulphate heptahydrate by the standard
spread plate method. The isolates were studied based on their cultural, morphological and
biochemical characteristics and were identified as strains of Bacillus azotoformans, Bacillus
megaterium, Micrococcus varians, Serratia marcescens HM1, Serratia marcescens HM2,
Proteus mirabilis, Aeromonas hydrophila HM3, Aeromonas hydrophila HM4, Aeromonas
caviae and Citrobacter spp. Further testing of the bacterial isolates in nutrient agar
supplemented with different concentrations (2, 4, 6, 8 and 10 mM) of hydrated zinc sulphate
showed that eight of the isolates (80%) exhibited resistance to Zn2+
concentration of 6 mM
and above. Co-resistance of four bacterial isolates to Pb2+
was studied and they showed high
resistance to the heavy metal with minimum inhibitory concentration (MIC) in the range of 2
to 8 mM. Out of the ten bacterial strains, seven (70%) exhibited resistance to several
antibiotics including amoxicillin, cotrimixazole, augmentin, nitrofurantoin and tetracycline,
and intermediate or full sensitivity to ofloxacin, nalidixilic acid and gentamicin. The bacterial
isolates obtained in the present study enrich studies on microbial diversity of zinc resistance
and could be exploited for biotechnological remediation of zinc, lead and other heavy metal
contaminated ecosystems.
Keywords: Wastewater, heavy metal, resistance, antibiotics, bacteria
1. Introduction
The metal coil surface coating industry includes any manufacturing facility engaged in the
surface coating of metal coil mainly; aluminum, galvanized steel, and zinc/aluminum, and
small quantities of brass (EPA, 1998). The coil coated metal is fabricated into end products
after it is coated, thus eliminating the need for post-assembly painting. Most coil coating
plants generate wastewater from wet section and quenching operations as well as clean-up
activities. Biological and physical/chemical treatment technologies and upstream waste
minimization activities are the building blocks in the development of industrial pollution

Isolation and characterization of zinc resistant bacteria from a coil coating industrial
wastewater treatment plant
International Journal of Environmental Sciences Volume 5 No.5, 2015
1031
control strategies. In Nigeria, despite the issuance of guidelines and standards that relate to
environmental pollution control by the Nigerian Federal Environmental Protection Agency
(FEPA, 1991), these regulations are not strictly enforced nor followed (Okereke, 2007).
Following the chemical and pharmaceutical industrial sector, the second most polluting are
the basic metal manufacturing facilities engaged in steel manufacturing, metal fabrication,
aluminum extrusion and related categories. Majority of these industries discharge their
partially treated or untreated effluents into the environment (Oketola and Osibanjo, (2011);
out of the 14 industries examined in the study, only 29% had effluent treatment plant which
was operational, 36% had no effluent treatment plant while the remaining 36% operate dry
process in which effluent treatment plant was not applicable. This dismal conventional
effluent analysis is comparable to the status of the United States’ coil coating industry in the
early 1980’s when approximately 15% of coil coating companies reportedly had no
wastewater treatment in place (EPA, 1980). According to that report, just about a third to
half of coil coating companies with wastewater treatment conducted some aspects of the
treatment process such as filtering to remove zinc and aluminum particles, oil skimming for
oil removal, lime precipitation of metals, pH adjustment, hexavalent chromium reduction,
filtration of total suspended solids, membrane processes or ion exchange systems to remove
dissolved salts from wastewater, clarification, sludge evaporation and reclamation (EPA,
1980). Consequently, regulated pollutants for coil coating industry include chromium,
cyanide, zinc, aluminum, cadmium, lead, nickel, selenium, tin, and mercury; oil and grease,
total suspended solids, and pH; nutrients and volatile organic compounds (VOCs) such as
ammonia, nitrogen, phosphorus, chloroethane, 1,1,1-trichloroethane, dichloroethane, and
dichloroethylene (EPA, 1980). Untreated wastewater when released to the rivers downstream
of point sources constitute an important health risk for the population using this water for
other purposes such as agricultural land irrigation, cleaning, bathing or even drinking,
whereby heavy metals and other hazardous chemicals often make their way into the food
chain (Echiegu and Liberty, 2013; Osho et al., 2010).
Introduction of high concentrations of heavy metals into the environment kills the majority of
the microflora, thereby creating selective pressure for the emergence of a few strains with
resistance to the metals. Such resistant strains participate in the process of self-recovery of
the contaminated habitat through a variety of mechanisms such as differences in uptake
and/or transport of the toxic metal while in other cases, the metal may be enzymatically
transformed by oxidation, reduction, methylation or demethylation into chemical species
which may be less toxic or more volatile than the parent compound (Nies, 1999). These
mechanisms are sometimes encoded in plasmid genes in close proximity with antibiotic-
resistance genes thus facilitating the transfer of toxic metal resistance from one cell to another
(Nageswaran et al., 2012) and cross resistance to both heavy metals and antibiotics (Devika
et al., 2013).
Although, heavy metal resistant bacteria have been demonstrated to exhibit high metal
biosorption or bioaccumulation capacity in the laboratory setting (Yilmaz, 2003; Ansari and
Malik, 2007; Bautista-Hernández, 2012) and some heavy metal resistant strains have been
successfully applied in remediating contaminated sites elsewhere in the developed world
(Canstein et al., 1999; Okino et al., 2002; Nakamura et al., 1999), similar efforts are
presently scanty in Nigeria (Odokuma and Akponah, 2010; Sanuth et al., 2010). The
principal aim of the present study was to assess the potential occurrence of Zn2+
resistant
bacteria from a coil coating industrial wastewater effluent, evaluate the maximum heavy
metal tolerance as well as determine co-resistance against antibiotics; all initial steps in
capacity building for biosorption/bioaccumulation studies.

1032
2. Materials and Methods
2.1 Study area and sample collection
Treated wastewater samples were collected from industrial wastewater treatment plant in a
coil coating company in Ota, the capital of the Ado-Odo/Ota Local Government Area of
Ogun State, Southwest, Nigeria. The coil coating facility is engaged in the production of a
variety of coated materials covering aluminum, galvanized steel and zinc/aluminum steel for
roofing applications. The wastewater treatment system receives wastewater from wet section
operations, quenching operations and clean-up activities. The following treatment activities
were in place at the treatment plant: oil skimming for oil removal, lime precipitation of
metals, clarification, sludge evaporation, sludge landfill or reclamation and effluent discharge.
Samples of wastewater from the treated effluent at the point of discharge to the environment
were aseptically collected in sterile screw-capped bottles and transported immediately to the
laboratory for analysis.
2.2 Enrichment, isolation and identification of metal resistant bacteria
For the isolation of heterotrophic bacteria, 1 ml of treated wastewater effluent was inoculated
into 9 ml of nutrient broth and incubated for 24 h at 370
C with shaking at 100 rpm. Ten-fold
serial dilutions of the overnight cultures were prepared. An aliquot (0.1 ml) of the diluted
samples was spread on sterile nutrient agar plates amended with 1 mM of zinc heptahydrate.
The plates were incubated at 370
C for 48 h. After the incubation period, the plates were
observed for growth on the media. The isolated and distinct colonies on the media were sub-
cultured repeatedly on the same media for purification. The purified isolates were grown in
nutrient agar slants 370
C for 24 h and kept in storage at 40
C. The isolates were identified on
the basis of their morphology and biochemical characteristics following the schemes
described (Madigan et al., 2009; Hemraj et al., 2013) and comparison with Bergey’s Manual
of Determinative Bacteriology (Holt et al., 1994).
2.3 Determination of maximum tolerated concentrations (MTCs) of heavy metals
The MTCs were determined for the bacterial isolates on nutrient agar medium in presence of
each of zinc and lead separately. The isolates were inoculated on nutrient agar plates
containing singly Zn2+
and Pb2+
at concentrations of 2, 4, 6, 8, 10 mM. The organisms were
incubated at 370
C for 72 h. The MTC was noted when the isolate failed to show growth on
the plates after the three days of incubation. All experimental set-ups were prepared in
duplicate.
2.4 Determination of antibiotic susceptibility
The bacterial isolates were tested for susceptibility to 8 different antibiotics by the disc
diffusion method on Mueller Hinton agar (Oyetibo et al., 2010). The antibiotics tested were
ofloxacin 5 µg (OFL), amoxicillin 20 µg (AMX), cotrimixazole 25 µg (COT), nitrofurantoin
300 µg (NIT), nalidixilic acid 30 µg (NAL), augmentin 30 µg (AUG), tetracycline 30 µg
(TET) and gentamicin 10 µg (GEN). The antibiotics discs were placed on Mueller Hinton
agar plates previously seeded with cell suspension with a turbidity of 0.5 McFarland
standards. The plates were incubated at 370
C for 24 h and observed for zones of inhibition.

1033
3. Results
Samples of treated wastewater from a coil coating industrial effluent examined in this study
contained heavy metal resistant bacteria. A total of 10 different, Gram negative (70%) and
Gram positive (30%), bacterial strains were isolated from the treated wastewater effluent
samples on nutrient agar – supplemented with 1 mM zinc sulphate heptahydrate by the
standard spread plate method. The Zn2+
resistant bacterial isolates were identified by
comparing their cellular and colonial morphological characteristics and the results from
biochemical tests with Bergey’s Manual of Determinative Bacteriology (Holt et al., 1994).
The results shown in Table 1 indicate the putative identity of the bacterial strains as follows:
Bacillus azotoformans, Bacillus megaterium, Micrococcus varians, Serratia marcescens
HM1, Serratia marcescens HM2, Proteus mirabilis, Aeromonas hydrophila HM3,
Aeromonas hydrophila HM4, Aeromonas caviae and Citrobacter spp.
Table 1: Zinc resistant bacterial isolates from treated wastewater effluent
Isolate
G
R
CM Cat Cit Ind MR VP Oxi Sta Glu Lac Man Putative Identity
A
+ Rods - + - + - + - - + + Bacillus
azotoformans
B
- Rods + - - + + - - + + + Serratia
marcescens HM1
C
+ Rods + + - + - - + + + + Bacillus
megaterium
D - Rods + + - + - - - + - + Proteus mirabilis
E
- Rods + + - + + - - + - + Serratia
marcescens HM2
F
- Rods + - - + - + - - - - Aeromonas
hydrophila HM3
G
- Rods + - - + - + - + - + Aeromonas
caviae
H
+ Cocci + - - + - - - + + - Micrococcus
varians
I
- Rods + - - + - + - + - - Aeromonas
hydrophila HM4
J - Rods + + + + - - - + + + Citrobacter spp.
GR: Gram reaction, CM: Cell Morphology, Cat: Catalase production, Cit: Citrate utilization,
Ind: Indole production, MR: Methyl Red, VP: Voges_Proskauer reaction, Oxi: Oxidase
activity, Sta: Starch hydrolysis, Glu: Glucose fermentation, Lac: Lactose fermentation, Man:
Mannitol fermentation, +: Positive, -: Negative
Maximum tolerance concentrations of Zn2+
were determined for each of the ten Zn2+
resistant
bacterial isolates by gradually increasing the concentrations of Zn2+
by 2 mM, on nutrient
agar until the strains failed to give colonies on plates. Table 2 presents the results of Zn2+
tolerance tests on nutrient agar. The results show a decline in the growth of the selected Zn2+
resistant bacterial isolates as the concentrations of Zn2+
increased. Strains of Serratia
marcescens HM1, Aeromonas caviae, Micrococcus varians and Aeromonas hydrophila HM4
were able to grow at up to 8 mM concentration of Zn2+
. Bacillus azotoformans, Proteus
mirabilis and Citrobacter spp. exhibited tolerance of Zn2+
at up to 6 mM; Bacillus
megaterium tolerated up to 4 mM while Aeromonas hydrophila HM3 strain did not grow at
any of the Zn2+
(2, 4, 6, 8 and 10 mM) concentrations tested.

1034
Table 2: Zinc tolerance test of bacterial isolates on Nutrient agar
Zinc concentrations in mM
Bacterial isolates 2 4 6 8 10
Bacillus azotoformans
Serratia marcescens HM1
Bacillus megaterium
Proteus mirabilis
Aeromonas hydrophila HM3
Aeromonas caviae
Micrococcus varians
Aeromonas hydrophila HM4
Citrobacter spp.
+++
+++
++
+++
++
-
+++
+++
+++
+++
++
++
++
++
++
-
+++
+++
+++
++
++
++
-
+
+
-
++
++
++
+
-
+
-
-
-
+
+
+
-
-
-
-
-
Key: -: No growth; +: Scanty growth; ++: Moderate growth; +++: Heavy growth
Four of the highly zinc resistant strains were further tested for growth in the presence of
increasing Pb2+
concentrations (2, 4, 6, 8 and 10 mM) on nutrient agar. Table 3 shows that
Serratia marcescens HM1, Aeromonas caviae and Citrobacter spp. exhibited tolerance for
Pb2+
at up to 8 mM while Micrococcus varians could only grow at 2 mM concentration of
this heavy metal.
Table 3: Lead tolerance test on selected bacterial isolates on Nutrient agar
Lead concentrations in mM
Bacterial isolates 2 4 6 8 10
Aeromonas caviae
Micrococcus varians
Citrobacter spp
++
+++
++
+++
+
+++
-
++
+
+++
++
+
++
++
-
-
-
Key: -: No growth; +: Scanty growth; ++: Moderate growth; +++: Heavy growth
The results of antibiotic sensitivity tests as shown in Table 4 indicate that seven (70%) of the
ten Zn2+
resistant bacterial strains exhibited resistance to several antibiotics including
amoxicillin, cotrimixazole, augmentin, nitrofurantoin and tetracycline, and intermediate or
full sensitivity to ofloxacin, nalidixilic acid and gentamicin. Both Aeromonas caviae and
Aeromonas hydrophila HM4 appeared to be resistant to most number of antibiotics (75%)
while Bacillus megaterium and Proteus mirabilis were resistant to three (37.5%) and
sensitive to five antibiotics. All ten zinc resistant bacterial isolates were sensitive to ofloxacin.

1035
Table 4: Antibiotic sensitivity pattern of zinc resistant bacteria from treated wastewater
effluent
Bacterial Isolates OFL AMX COT NIT NAL AUG TET GEN
Bacillus azotoformans S R R R S R R I
Serratia marcescens HM1 S R R R S R R I
Bacillus megaterium S R R S S R S I
Proteus mirabilis S R R S S R S I
Serratia marcescens HM2 S R R R S R R I
Aeromonas hydrophila
HM3
S R R R I R R I
Aeromonas caviae S R R R S R R R
Micrococcus varians S R R R R R S S
Aeromonas hydrophila
HM4
S R R R I R R R
Citrobacter spp. S R R R I S R I
Key: OFL = 5 µg ofloxacin, AMX = 20 µg amoxicillin, COT = 25 µg cotrimixazole, NIT =
300 µg nitrofurantoin, NAL = 30 µg nalidixilic acid, AUG = 30 µg augmentin, TET = 30 µg
tetracycline, GEN = 10 µg gentamicin. S = Susceptible to antibiotic effect, R = Resistant to
antibiotic effect, I: Intermediate
4. Discussion
This study was carried out to assess the incidence of Zn2+
resistant bacteria from a coil
coating industrial wastewater treatment plant in Ota, Southwest Nigeria. Ten Zn2+
resistant
bacterial isolates recovered from the treated wastewater samples were putatively identified as
strains of Bacillus azotoformans, Bacillus megaterium, Micrococcus varians, Serratia
marcescens HM1, Serratia marcescens HM2, Proteus mirabilis, Aeromonas hydrophila HM3,
Aeromonas hydrophila HM4, Aeromonas caviae and Citrobacter spp on the basis of their
cultural, morphological and biochemical characteristics. Similar findings of occurrence of
Zn2+
metal resistant bacteria in contaminated soils, wastewater effluents, river water and fish
have been reported (Ahemad and Malik, 2012; Bhadra et al., 2007; Akinbowale et al., 2007;
Jackson et al., 2012; Mgbemena et al., 2012). Ahemad and Malik (2012) characterized and
identified five Zn2+
resistant Bacillus spp. from Indian agricultural soils irrigated with metal
polluted wastewater. Aeromonas species, Proteus species and Micrococcus species isolated
from Otamiri River, Imo State, Nigeria were demonstrated by Mgbemena et al. (2012) to
tolerate the presence of Zn2+
at high concentrations. Jafarzade et al. (2012) also described
Serratia spp. isolated from a marine environment in Malaysia that was highly resistant to
Zn2+
. Some members of the genus Citrobacter isolated from heavy metal-contaminated sites
have been found with the ability to resist and accumulate Zn2+
(Jeong and Macaskie, 1995).
Microbial exposure to heavy metals selects and maintains microbial variants able to tolerate
the harmful effects of metals. Varied and efficient metal resistance mechanisms have been

1036
identified in diverse species of bacteria (Nies, 1999; Dopson et al., 2003; Issazadeh, 2013).
Some of the Zn2+
resistant bacterial isolates characterized in this study also showed resistance
to Pb2+
. In the study by Ahemad and Malik (2012), the zinc resistant bacteria (Pseudomonas
isolate SN7, Pseudomonas isolate SN28 and Pseudomonas isolate SN30) exhibited co-
resistance against Cu2+
, Hg2+
, Cd2+
, Ni2+
, Pb2+
, Cr3+
and Cr6+
in addition to Zn2+
. Efflux
transporters belonging to the P1B-type subfamily of ATPases are thought to play a key role
in heavy metal homeostasis of essential metals such as Cu2+
, Co2+
, and Zn2+
, as well as
mediating resistance to toxic metals Pb2+
, Cd2+
and Ag2+
(Axelsen and Palmgren,1998). An
alternative resistance mechanism of Zn2+
and Pb2+
dependent upon metabolic energy of
microorganisms is the bioaccumulation of both heavy metals (Augusto da Costa and Duta,
2001). It remains to be determined which mechanism(s) accounted for bacterial resistance to
both zinc and lead in the bacterial isolates in this study.
The MTCs for Zn2+
and Pb2+
in this study ranged, respectively, between 1 and 8 mM, and 2
and 8mM, depending on the bacterial strains. The methodologies employed for the
determination of maximum tolerated concentrations of heavy metals for resistant bacteria
have been inconsistent from study to study; while some have used liquid media (Hassen et al.,
1998), most have conducted the determinations in solid media (Kermanshahi et al., 2007;
Bautista-Hernández et al., 2012; Xu et al., 2014) as was done in the present study. It is
generally considered that heavy metals are more toxic in liquid than in solid media due to
more dispersion in the culture (Haferburg et al., 2007).
The Zn2+
and Pb2+
resistant bacterial isolates identified in this study may be considered to fall
into three categories on the basis of tolerance to these metals; high (8 mM), medium (4-6
mM) and low at 1-2 mM concentrations. Thus Serratia marcescens HM1 and Aeromonas
caviae are considered to be highly tolerant of Zn2+
or Pb2+
given their growth on solid media
at up to 8 mM concentration of either metal. Micrococcus varians and Aeromonas hydrophila
HM4 also demonstrated high tolerance to Zn2+
at 8 mM. Bacillus azotoformans, Bacillus
megaterium, Proteus mirabilis and Citrobacter spp. exhibited moderate tolerance to Zn2+
at 4
to 6 mM concentrations. Aeromonas hydrophila HM3 strain grew on solid media at 1 mM
Zn2+
concentration. Interestingly, Citrobacter spp. exhibited high tolerance for Pb2+
at up to 8
mM while Micrococcus varians could only grow at 2 mM concentration of this heavy metal.
The range of maximum tolerated concentrations of Zn2+
metal observed for Serratia
marcescens HM1, Aeromonas caviae, Micrococcus varians, Aeromonas hydrophila HM4 and
Citrobacter spp. are comparable to the minimum inhibitory concentrations of up to 10 mM
Zn2+
ions previously reported for Pseudomonas aeruginosa HMRI and P. aureginosa HMR2
(Bhojiya and Joshi, 2012) or P. putida strain 06909 (Lee et al., 2001). Similarly, the
maximum concentration of Pb2+
tolerated by Serratia marcescens HM1, Aeromonas caviae
and Citrobacter spp compares favorably to the MTC of up to 10 mM for a Pseudomonas sp.
(Owolabi and Hekeu, 2014). Like some isolates in this study; Aeromonas hydrophila HM3
and Micrococcus varians, Devika et al. (2013) have demonstrated the minimum inhibitory
concentration of Zn2+
and Pb2+
ions up to 2-3 mM for an Enterobacter sp.
In the present study, seven out of the ten (70%) heavy metal resistant bacterial strains
exhibited resistance to several antibiotics including amoxicillin, cotrimixazole, augmentin,
nitrofurantoin and tetracycline, and intermediate or full sensitivity to ofloxacin, nalidixilic
acid and gentamicin. Our data indicate co-occurrence of heavy metal resistance with
antibiotic resistance. Similar results have been reported previously (Bahig et al., 2008;
Oyetibo et al., 2010; Owolabi and Hekeu, 2014; Jafarzade et al., 2014). In the study by Bahig

1037
et al. (2008), 33% to 60% of the heavy metal resistant bacterial isolates from soils irrigated
with canal water or wastewater showed resistance to ampicillin, kanamycin and tetracycline.
Five heavy metal resistant strains; Pseudomonas aeruginosa, Actinomyces turicensis,
Acinetobacter junni, Nocardia sp., and Micrococcus sp., isolated from soils obtained from
Ikeja industrial estate, Lagos, Nigeria resisted all the 18 antibiotics evaluated (Oyetibo et al.,
2010). In the study reported by Jafarzade et al. (2012), three strains of Serratia spp. isolated
from marine environment were highly resistant to penicillin, ampicillin and tetracycline but
sensitive to nalidixic acid, streptomycin, kanamycin and gentamicin.
Samples of treated wastewater from a coil coating industrial effluent in Ota, examined in this
study contained diverse bacterial isolates that are moderately to highly resistant to heavy
metals and antibiotics. Dual resistant organisms would have the capacity to compete well
with antibiotic-producing flora in the polluted environment and may also help to overcome
the inhibition that heavy metals exert on the biodegradation of organic pollutants (Sandrin
and Maier, 2003). Further research is warranted at the molecular level to determine the
mechanism of these dual properties and their close association with one another. The
biosorption capacity of these bacterial strains remains to be assessed experimentally as an
important requisite for their potential use in practical bioremediation of heavy metal
accumulation in wastewater and soils.
5. Acknowledgement
The authors would like to acknowledge the Management of Covenant University for
providing research infrastructure and support systems that made this work possible.
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