ISOLATION AND CHARACTERIZATION OF BACTERIAL STRAINS THAT ARE
RESISTANT TO NICKEL, COBALT AND OTHER HEAVY METAL
Norashikin Bt. Badaruddin Afandi (19401)
A thesis submitted in fulfillment of the requirements for the degree of
Bachelor of Science with Honours
(Resource Biotechnology)
Faculty of Resource Science and Technology (FRST)
UNIVERSITI MALAYSIA SARAWAK (UNIMAS)
2010
I
ACKNOWLEDGEMENT
First of all, I would like to give my sincere to thank Prof. Dr. Mohd Azib Bin Salleh for
his worthy guidance and valuable supervision of this research project.
I owe special thanks to all my lecturers, laboratory assistants, and all my friends,
especially Mohd Taufik Bin A. Malek, Intan Nurliyana Binti Omar, Mazidatul Ashiqeen
Balqiah Binti Mohamad Lazim, Oliver Swenson Ak Ragib, and Jakaria Bin Tuan Haji
Rambeli for their help, support and the relaxed atmosphere throughout this final year
project completition.
Finally, I want to express my endless gratitude to my parents and my siblings for their
continuous moral support.
II
TABLE OF CONTENTS
ACKNOWLEDGEMENT ……...………………………..…...…………………..
I
TABLE OF CONTENTS ……………….…………………………...…………... II
LIST OF ABBREVIATIONS ……………..………………….……...….……….
IV
LIST OF TABLES ………………………….……………….………….….……..
LIST OF FIGURES ………………………………………………………………
V
VI
ABSTRACT …………………………………………………………...…………..
1
1.0 INTRODUCTION AND OBJECTIVE ….…..……
2
2.0 LITERATURE REVIEW ………………………....
2.1 Heavy Metal Background …..………………………..…….……......
2.1.1 Presence of Heavy Metals in Environment ……….…...….....
2.1.2 Nickel – Cobalt …………….………………………...………
2.2 Bacterial Tolerance against Heavy Metal ………...….………….....
2.3 Basic Mechanism of Tolerance ……………...………...……………
2.3.1 Metal Tolerance Mechanism ……………….………………..
2.4 Genetic Studies …………………………………………...………….
4
4
4
5
6
7
7
8
3.0 MATERIALS AND METHOD ……………………
3.1 Sample Collection ………………………………………………........
3.2 Growth Medium ……………………….…………………………….
3.3 Heavy Metal Stock Solutions ………….……………………..….......
3.4 Isolation of Bacterial Strains ………….…………………………….
3.4.1 Storage of Bacterial Isolates ….……………………………...
3.4.2 Master Plates Preparation …..………………………………..
3.5 Measurement Level of Bacterial Resistant ……..………………….
3.5.1 Ni2+
and Co2+
Heavy metal ……….….………………………
3.5.2 Multiple Heavy Metals ………………………………………
10
10
10
12
13
13
13
14
14
14
III
3.6 Identification of Bacterial Strains …………….…………………….
3.6.1 Gram Staining …………………..……………………………….
3.6.2 Biochemical Testing …………………………………………….
3.7 ‘Miniprep’ Plasmid Isolation and AGE ……………….…………...
15
15
15
15
4.0 RESULTS ………...…...………………….…………
4.1 Isolation and Identification of Bacterial Isolates …....……..............
4.2 Patterns of Heavy Metal-Resistance and their Frequency...….…...
4.3 Measurement Level of Resistant ………...………..…………...........
4.4 Identification of Heavy Metal-Resistance Bacterial Isolates ……...
4.5 Occurrence of Plasmids in Bacterial Isolates.…………...……...….
16
16
16
19
20
20
5.0 DISCUSSION …………………………………...…..
23
6.0 CONCLUSION ………………….…………………. 27
REFERENCES ………………………………………………………………….. 29
IV
LIST OF ABBREVIATIONS
µg microgram
Co cobalt
Cu copper
cm centimetre
cnr cobalt-nickel resistance system
cre cobalt resistant system
DNA deoxyribonucleic acid
Fe iron
g gram
Hg mercury
LB Luria-Bertani medium
MHA Mueller Hinton agar
ml millimetre
MP Master Plate
NA Nutrient agar
NB Nutrient Broth
ng nanogram
Ni nickel
nre nickel resistant system
⁰C degree celcius
SDS sodium dodecyl sulphate
sp. species
Zn zinc
V
LIST OF TABLES
Table 3.1 Heavy metal salts used in this study. 12
Table 4.1 Morphology and Gram reactions of multiple-resistant
bacterial isolation.
17
Table 4.2 Heavy metal-resistance profiles of bacterial isolates. 18
Table 4.3 Determination of the levels of resistance. 19
Table 4.4 Biochemical characteristics of the five bacterial isolates. 21
VI
LIST OF FIGURES
Figure 3.1 The location of sampling sites at Pending, Kuching,
Sarawak.
11
Figure 4.1 Agarose gel electrophoresis to detect the presence of
plasmids.
22
1
Isolation and Characterization of Bacterial Strains that are Resistant to Nickel,
Cobalt and other Heavy Metal
Norashikin Bt. Badaruddin Afandi
Biotechnology Resource Programme
Faculty of Resource Science and Technology
Universiti Malaysia Sarawak
ABSTRACT
A total of 500 bacterial strains were isolated from soil samples collected near the non-ferrous industrial
sites (industrial effluent) at Pending, Sarawak. Two hundred and thirty bacterial isolates were found to be
resistance against Co, Ni, Cu, Zn, Hg and Fe in different patterns of resistance. Heavy metal-resistance
pattern to CoR
and NiR showed the highest percentage (36%). The levels of resistance of bacterial isolates
were determined by testing against various concentrations of heavy metals. Mercury was the most toxic
metal which inhibits the growth of bacterial isolates at 15µg/ml. The toxicity order of the metals were Hg
> Co > Cu > Ni > Fe > Zn. Five bacterial isolates were selected for further analysis based on their multiple
patterns of resistance (CoR
NiR
CuR
FeR
ZnR
HgR). The morphological and biochemical characteristics of
five bacterial isolates showed that they were putative strains of the genus Ralstonia Eutropha
(Alcaligenes), Pseudomonas aeruginosa, Klebsiella oxytoca. DNA analysis of all the five representative
isolates which showed multiple tolerances of heavy metals did not reveal the presence of any plasmid.
Key words: Bacterial, heavy metal, resistance, characteristics, DNA analysis.
ABSTRAK
Sebanyak 500 bakteria diasingkan daripada sampel tanah yang diambil berhampiran lokasi industri
bukan ferus (sisa industri) di Pending, Sarawak. 230 bakteria yang telah diasingkan dikenalpasti rintang
terhadap logam Co, Ni, Cu, Zn, Hg dan Fe dalam pola kerintangan yang berbeza. Pola kerintangan
logam berat CoR dan Ni
R menunjukkan peratusan yang tertinggi (36%). Tahap kerintangan bakteria
tersebut ditentukan dengan menggunakan kepekatan logam berat yang bebeza. Merkuri merupakan logam
paling toksik yang menyekat pertumbuhan bakteria pada 15μg/ml. Urutan tahap toksik logam yang
dikenalpasti adalah Hg> Co> Cu> Ni> Fe> Zn. Lima bakteria tertentu sebagai wakil yang dipilih
berdasarkan pola kerintangan bacteria tersebut (CoR
NiR
CuR
FeR
ZnR
HgR). Ciri-ciri lima bakteria yang
bersesuaian dianggap milik tiga ‘genus’ iaitu Ralstonia eutropha (Alcaligenes), Pseudomonas aeruginosa,
Klebsiella oxytoca. Analisis DNA yang dilakukan ke atas kesemua wakil lima bacteria tersebut yang
menunjukkan kerintangan terhadap beberapa logam berat menunjukkan tiada kewujudan plasmid.
Kata kunci: Bakteria, logam berat, kerintangan, ciri-ciri, analisis DNA.
2
1.0 INTRODUCTION
Soil contains a variety of microorganisms including bacteria which are essential for the
maintenance of nutrients and geochemical cycles (carbon, nitrogen, sulphur and
phosphorus cycle) (Kummerer, 2004). Nowadays indiscriminate and uncontrolled
discharge of metal-contaminated industrial effluent in the environment has become an
issue of major concern. Bacteria resistant to nickel and cobalt have been isolated from
ecosystems polluted by heavy metals, such as agricultural, industrial waste, (Jian Tian et
al., 2007).
Some bacteria (Ralstonia sp. and Alcaligenes sp.) have evolved mechanisms
that regulate metal ion accumulation to detoxify heavy metals and some even use them
for respiration (Grass, 2000). Microbial survival in polluted soils depends on intrinsic
biochemical and structural properties, as well as physiological and genetic adaptation
(morphological changes in cell as well as environmental modification) (Pradipta, 2006).
Microbes may play a major role in the biogeochemical cycling of toxic heavy
metals and in cleaning up or remediating metal-contaminated environments (Kummerer,
2004). Although some heavy metals are essential trace elements, most can be, at high
concentrations, toxic to all branches of life, including microbes, by forming complex
compounds within the cell. Most mechanisms studied involve the efflux of metal ions
outside the cell, and genes for this general type of mechanism have been found on both
chromosomes and plasmids (Indu, 2006).
3
Heavy metals are increasingly found in microbial habitats due to natural and
industrial processes. Hence, microbes have evolved several mechanisms to tolerate the
presence of heavy metals (by efflux, complexation, or reduction of metal ions) or to use
them as terminal electron acceptors in anaerobic respiration.
Thus far, tolerance mechanisms for metals such as copper, zinc, arsenic,
chromium, cadmium, and nickel have been identified and described in detail. The
toxicity of heavy metals to bacteria, with particular reference to metal forms and species,
has been reviewed (Elizabeth, 2003). Many have speculated and have even shown that a
correlation exists between metal tolerance resistance in bacteria because of the
likelihood that resistance genes to heavy metals may be located closely together on the
same plasmid in bacteria and are thus more likely to be transferred together in the
environment (Mergeay, 2000). Latest reports on heavy metal-resistant studies found
those bacteria which are Gram -ve bacteria (Ralstonia sp., Alcaligenes sp., Pseudomonas
spp.) and Gram +ve bacteria (Streptomyces sp.) (Pradipta, 2006).
The present project was conducted, with the objective of isolating and
characterizing heavy metal-resistant bacteria from soil environment. Besides, this study
was also aimed at isolating plasmid from the metal-resistant bacterial strains.
4
2.0 LITERATURE REVIEW
2.1. Heavy Metal Background
There are approximately sixty-five elements, which may be termed as ‘heavy metal’ as
they exhibit metallic properties with a density above 5 g/cm3 (Pradipta, 2006). Thus, the
translations elements from vanadium (V) [but not scandium (Sc) and titanium (Ti)] to
the half metal arsenic (As), from zirconium (Zr) [but not yttrium (Y)] to antimony (Sb),
from lanthanum (La) to polonium (Po), the lanthanides and the actinides can be referred
to as heavy metals (Nies and D.H., 1999). In form of cation, some of the heavy metals
are essential (cobalt, chromium, nickel, zinc, copper, vanadium and tungsten) which are
required by the organisms as micro nutrients (trace elements) at 10-9
M concentrations.
However, at 10-3
M concentrations, both metals which essential and with no essential
biological functions (arsenic, silver, cadmium, antimony, lead, mercury and uranium)
lead to toxic effects (Indu, 2006).
2.1.1 Presence of Heavy Metal in Environment
In natural environments, heavy metals exist throughout the world due to their use in
industrial countries for a variety of applications like agricultural, industrial waste,
municipal waste disposal and mining. Industrial with manufacturing textile, allied
chemicals, electroplating, batteries, paints, plastics, and petrochemicals have been
reported to contain high concentrations of various heavy metals in those industrial
environment such cadmium, chromium, arsenic, cobalt, nickel, copper, mercury, and
lead (Fakayode and Onianwa 2002; Oyeyiola et al. 2006).
5
2.1.2 Nickel-Cobalt
Cobalt and nickel are used in the production of steel and alloys which are the mains
components used in manufacture of coins, magnets, household utensils, steels, batteries,
electroplating and production of blue and green pigments (Carnes, 2009). Nickel toxicity
is comparable to cobalt but its toxic effect on humans is better documented, up to 20% of
the populations in industrially developed countries have positive results in epidermal
testing (Savolainen, 1996).
Many industries such as electroplating, paint, pigments, batteries, and gas
turbines, discharge aqueous effluents containing relatively high levels of nickel and cobalt.
Trace elements such as chromium, lead, and nickel, have been detected from industrial
effluents collected in and around industrial areas (Sivakumar et al., 2001).
Both nickel and cobalt are required as an essential cofactor in several bacterial
enzymes which carry out a variety of metabolic functions (Mulrooney and Hausinger, 2003),
but it disrupts these processes when it is present in excess (Babich and Stotzky, 1983).
6
2.2 Bacterial Tolerance against Heavy Metals
The quantity of heavy metal released in the environment has become rapidly expansion
which cause evolved stage found in wide range of microbial groups and species with
genetic or physiological adaptation under extreme or stress environment by having the
ability to survive and grow in the presence of relatively high metal concentration in
several habitats (Bruins et al., 2000).
Recent studies have shown the microbes were found to belong to contain
genes. These bacteria are mainly Ralstonia eutropha CH34, Alcaligenes denitrificans 4a-
2, Alcaligenes xylosoxydans 31A, Ralstonia eutropha KTO2, Klebsiella oxytoca CCUG
15788, Hafnia alvei 5-5, and Escherichia coli (resistant to nickel and cobalt) (Jian Tian
et al., 2007). The mechanisms of nickel and cobalt resistance in bacteria are due to the
action of an operon-encoded, energy-dependent specific efflux system that pumps the
cation from the cell, thereby lowering the intracellular concentration of the toxic metal
(Park et al., 2004).
Other bacteria recently found are Saccharomyces cerevisiae (resistant to zinc,
manganese, copper, iron and chromium), Pseudomonas sp. (resistant to chromium and
uranium), Enterobacter sp. (resistant to silver), Citrobacter sp., and Staphylococcus
aureus (resistant to arsenic and cadmium).
7
2.3 Basic Mechanism of Tolerance
There are four mechanisms of bacterial metal-resistant. 1) Keeping the metal ions out of
the cell (reduced uptake) (Grass et al., 2000). 2) Highly-specific efflux pumping (e.g. the
mechanism of nickel resistance in bacteria is due to the action of an operon-encoded,
energy-dependent specific efflux system that pumps the cation from the cell, thereby
lowering the intracellular concentration of the toxic metal (Jian Tian, 2007). 3) Intra- or
extracellular sequestration by specific mineral-ion binding components (e.g.
metallothioneins). 4) Enzymatic detoxification (oxydoreductions), which converts a
more toxic ion to a less toxic one. Quite often, several different resistance mechanisms
for a same metal may be found among the same microbial species (Zgurskaya and
Nikaido, 2000).
2.3.1 Metal Tolerance Mechanisms
Microorganisms have acquired a variety of mechanisms for adaptation to the presence of
toxic heavy metals. Among the various adaptation mechanisms, metal sorption, uptake,
mineralization, and accumulation, extracellular precipitation and enzymatic oxidation or
reduction to a less toxic form, and efflux of heavy metals from the cell has been reported
(Mergeay, 1991; Hughes and Poole, 1991; Nies, 1992; Urrutia and Beveridge, 1993;
Joshi-Tope and Francis, 1995).
In high concentrations, heavy metal ions react to form toxic compounds in
cells (Nies, 1999). Some heavy metals are necessary for enzymatic functions and
bacterial growth so, uptake mechanisms exist that allow for the entrance of metal ions
into the cell.
8
There are two general uptake systems which are quick and unspecific, driven
by a chemiosmotic gradient across the cell membrane and thus requiring no ATP, and
the other is slower and more substrate-specific, driven by energy from ATP hydrolysis.
While the first mechanism is more energy efficient, it results in an influx of a wider
variety of heavy metals, and when these metals are present in high concentrations, they
are more likely to have toxic effects once inside the cell (Nies and Silver, 1995).
In order to survive under metal-stressed conditions, bacteria have evolved
several types of mechanisms to tolerate the uptake of heavy metal ions. These
mechanisms include the efflux of metal ions outside the cell, accumulation and
complexation of the metal ions inside the cell, and reduction of the heavy metal ions to a
less toxic state (Nies, 1999).
2.4 Genetic Studies
Microorganisms possess mechanisms that regulate metal ion accumulation to avoid
heavy metal toxicity. Many species of bacteria have genes that coded and control
resistances to specific toxic heavy metals located on extra chromosomal elements of
DNA molecules (plasmid) (Kummerer, 2004).
For examples Ralstonia sp. strain CH34 is resistant to nickel and cobalt
cations. Resistance is mediated by the cnr determinant located on plasmid pMOL28 due
to an energy-dependent efflux system driven by a chemo-osmotic proton-antiporter
system (Taghvi et al., 2001). The cnr determinant is composed of at least six genes,
encoding products with regulatory functions (cnrY, cnrX, and cnrH) or the subunits of
the Co2+/
Ni2+
efflux pump (cnrC, cnrB, and cnrA) (Grass G et al., 2000).
9
The observations that metal resistance determinants are located most
frequently on plasmid and transposons have led to suggestions that these determinants
are probably spread by horizontal transfer. Such genetic systems are useful tools to
investigate the nature and extent of horizontal transfer of adaptive genes across natural
bacterial populations (Silver, 1992).
Broad-host-range expression of ncc-nre was recently confirmed by (Dong et
al., 1998) who found ncc-nre-based Ni resistance in Comamonas sp., Sphingobacterium
sp., flavobacteria sp., and even Gram-positive bacteria related to Arthrobacter sp. For
example, several nickel resistance determinants have been identified in Ralstonia
eutropha (Alcaligenes eutrophus) strains isolated from different biotopes heavily
polluted with heavy metals. Resistance to Cd2+
, Zn2+
and Co2+
has been shown to be
located on a czc operon of the plasmid pMOL30 (240 kb). Some report had a similar
observed in a wide variety of bacteria, especially in gram negative bacteria (Poole, 2002)
such as Pseudomonas spp., Ralstonia metallidurans and Enterobacter cloacae.
10
CHAPTER THREE
MATERIALS AND METHOD
3.1 Sample Collection
Soil samples were collected near the non-ferrous industrial sites (industrial effluent) in
the Pending Division (refer to Figure 3.1). Sterile digging tools were used to collect soil
samples in range of 10 to 20cm below the soil surface. The samples were placed in
sterile polyethylene bags. The bags which contained soil samples were labelled first,
then were placed in Laboratory of Molecular Genetic at FRST by stored the samples at
4⁰C until need for further extended used.
3.2 Growth Media
Nutrient agar (NA) and Müeller-Hinton agar (MHA) is purchased from Oxoid (UK),
while Luria-Bertani (LB) broth is ordered from Fluka (Switzerland). All growth media
were sterilized by autoclaved at 121⁰C. All heavy metal compounds used were
purchased from Ajax Chemicals (Laboratory UNILAB Reagent), Australia. Sodium
dodecyl sulphate (SDS) was obtained from BHD Laboratories Supplies (UK). SDS
solution (10% (w/v)) was prepared in ultra-pure water, and then sterilized. A fresh
solution was prepared for every experiment and was added to double-strength LB broth
(1:1) prior to use.
11
Figure 3.1 The location of sampling site at Pending, Kuching, Sarawak.
Location
of sample
collection
12
3.3 Heavy Metal Stock Solution
Heavy metals salts solutions will be prepared by diluting the appropriate weight of
metals salts in distilled water and sterilized by autoclaving at 121ºC for 15 minutes.
Heavy metal stock solutions is prepared due to the analytical grades of metal salts by
dissolved the respective heavy metal salts (refer to Table 3.1) in ultra-pure water. The
heavy metals stock concentration determined based on the solubility of the heavy metals
salts in water and their respective working concentrations. The proper volumes of heavy
metal stock solutions will be added to Müeller-Hinton agar (MHA) chilled to 55ºC at a
predetermined volume (100 ml, occasionally) to produce the desired final concentration.
Table 3.1 Heavy metal salts used in this study
Heavy Metal Salts Heavy Metal Cations Stock
Concentrations
Working
Concentrations
(µg/ml)
CoCl2. 6 H2O Cobalt, Co2+
200 mg/ml 50 – 1400
FeSO4. 7H2O Iron, Fe2+
400 mg/ml 50 – 2000
Ni (NO3)2. 6H2O Nickel, Ni2+
200 mg/ml 50 – 1600
ZnSO4. 7H2O Zinc, Zn+
400 mg/ml 50 – 2000
HgCl2 Mercury, Hg2+
15 mg/ml 15
CuSO4. 5H2O Copper, Cu2+
200 mg/ml 50 – 1600
13
3.4 Isolation of Bacterial Strains
The time interval between sampling and bacterial isolation can not exceed two weeks.
The original tube contains 1g of fresh soil sample with 1 ml distilled water. A 10-fold up
to 10-7
serial dilution is done by adding 0.1ml solution to saline solution (0.9ml) in each
tube. Then, the aliquot samples are transferred (0.1ml) from the 10-5
to 10-7
dilutions and
spread on nutrient agar (NA) plates. Spread plate technique was applied thus; three
replicates of NA plates are prepared for each dilution and incubate the plates at 30⁰C for
24 hours. The colonies are selected randomly with different morphological appearance
from that culture plates. Purified is done by further sub-cultured in the same media.
3.4.1 Storage of Bacterial Isolates
The well-defined isolated colonies were picked up on the basis of colony morphological
characteristics and transferred to nutrient agar and preserved in 20% (v/v) glycerol at -
20⁰C or -80⁰C (refrigerator 4℃).
3.4.2 Master Plates Preparation
500 of bacterial colonies that recovered on NA plates are picked then and spotted on a
master plate (MP). The colonies are selected randomly with different morphological
appearance from that original NA culture plates. The master plates are incubated at 37⁰C
overnight.
14
3.5 Measurement Level of Bacterial Resistance
3.5.1 Ni2+
and Co2+
Heavy Metal
The bacterial colonies on master plates are picked and spotted on the MHA plates which
supplied with nickel and cobalt heavy metal salts at various concentrations (refer to
Table 3.1). There are also bacteria control (ATCC Escherichia Coli 25922 sp.) that were
obtained from the stock collection of the Molecular Genetic Laboratory, Department of
Molecular Biology, Faculty of Resource Science and Technology, Universiti Malaysia
Sarawak (UNIMAS) which spotted on each of those MHA plates. Incubate the MHA
plates at 27⁰C overnight. The bacterial colonies that grew on the highest concentration of
Nickel and Cobalt metal supplements or high level with sensitive of bacteria control are
identified.
3.5.2 Multiple Heavy Metals
The identified colonies are referred back to the master plates. Thus, the identified
bacterial on the master plates are picked and streak on MHA plates which supplied with
different types of heavy metal salts at various concentrations (refer to Table 3.1).
There are also bacteria control (ATCC Escherichia Coli 25922 sp.) spotted on
each of those MHA plates. Incubate the MHA plates at 27⁰C overnight. The bacterial
colonies that are grown on the highest concentration of different types of heavy metal
supplements or high level with sensitive of bacteria control are identified. Those
identified bacterial colonies then, are isolated and re-streaked on new master plates and
incubated at 27⁰C overnight.
15
3.6 Identification of Bacterial Strains
3.6.1 Gram Staining
Staining are carried out by standard procedure of Gram Staining (Duguid, 1989). The
slides are observed under oil immersion used light microscopy (100 x magnifications) by
examining Gram reaction and its morphological appearances such as colour and the
shape of bacterial colony. Those bacterial colonies are identified up to their genus level,
according to diagnostics tables of Bergey’s Manual of Systematic Bacteriology (Krieg
and Holt, 1984).
3.6.2 Biochemical Testing
A few selected bacterial resistant strains were tested for a number of biochemical
characteristics, such as methyl red - Voges proskauer test, citrate utilization and catalase
test as described by Grimont and Grimont (1992).
3.7 ‘Miniprep’ Plasmid Isolation and AGE
The standard protocol of plasmid isolation is carried out by used a modified alkaline
lysis method (‘miniprep’) as described by Birnboim and Doly (1979). The best five
representatives of isolated plasmids were run used agarose gel electrophoresis (AGE)
according to standard procedure (Maniatis et al., 1989). The size estimates of the
isolated plasmids were obtained by comparing their relative mobilities on agarose gel
with standard molecular weight DNA marker (1kb). The plasmid DNA were visualize
under UV transilluminator.
16
CHAPTER FOUR
RESULTS
4.1 Isolation and Identification of Bacterial Isolates
A total of 500 bacterial strains isolates were isolated from sample collected at the non-
ferrous industrial sites (industrial effluent) in the Pending Division, Sarawak. Based on
the preliminary morphological examination of bacterial strains on NA, most of the
bacterial isolates revealed formed yellowish, entire and circular colonies. Some isolates
form white or cream-colored colonies; others showed the presence of pink or orange
pigments. Besides, microscopic analyses showed that most isolates were rod-shaped
Gram negative bacteria. Details of selected isolated strains for cell morphology and
Gram reaction are summarizing in Table 4.1.
4.2 Patterns of Heavy Metal Resistance and their Frequencies
Out of 500 isolates, 230 isolates were found to be resistant to one or more pattern of
heavy metals. Generally, the most frequently occurred among bacterial isolates were
resistance to nickel (47.5 %), followed by cobalt (31.25 %), copper (8.75 %), zinc (6.25
%), iron (3.75 %) and mercury (2.5 %) resistances. Thirty heavy metal-resistance
patterns (single and multiple resistances) were observed. Five isolates showed multiple
resistances to all six heavy metal screened, 16 showed resistance to five heavy metal, 15
showed resistance to four heavy metal, 16 showed resistance to three heavy metal and 90
showed resistance to two heavy metal. While the remaining isolates showed resistance to
only one heavy metal. Table 4.2 has summarizing the different patterns and frequencies
of the heavy metal-resistance.
17
Table 4.1 Morphology and Gram-reaction of multiple-resistant bacterial isolates
MP Bacteria Color & Shape Gram
MP 2 (42) CoR
NiR
CuR
FeR
ZnR
HgR
Pink (Rod) - ve
MP 3 (34) CoR
NiR
CuR
FeR
ZnR
HgR
Pink (Rod) - ve
MP 3 (45) CoR
NiR
CuR
FeR
ZnR
HgR
Purple (Cocci) + ve
MP 7 (30) CoR
CuR
FeR
ZnR
Purple (Cocci) + ve
MP 8 (44) CoR
NiR
CuR
FeR
ZnR
HgR Pink (Rod) - ve
MP 9 (26) NiR
CuR
FeR
ZnR
HgR Pink (Cocci) - ve
MP 10 (22) CoR
NiR
CuR
FeR
HgR Purple (Rod) + ve
MP 11 (3) CoR
NiR
CuR
FeR
ZnR
Pink (Cocci) - ve
MP 12 (45) CoR
NiR
CuR
FeR
Pink (Cocci) - ve
MP 14 (9) CoR
NiR
CuR
FeR
ZnR
HgR Pink (Rod) - ve
Abbreviations: Co – cobalt; Ni – nickel, Cu – copper, Fe – Iron, Zn – zinc,
Hg – mercury, R – resistance phenotype,
S – sensitive phenotype, MP – master plate,
+ ve – Gram positive bacteria, - ve – Gram negative bacteria
