J. Env. Bio-Sci., 2015: Vol.

29 (2):533-538
(533) ISSN 0973-6913 (Print), ISSN 0976-3384 (On Line)

PREVALENCE OF HEAVY METAL RESISTANT BACTERIA IN SAMPLES FROM

SEWAGE TREATMENT PLANTS AND COMMON EFFLUENTS TREATMENT PLANTS
P. Katyal*1, P. P. Sahota1, D. Singh2 and R. Sikka2
1
Department of Microbiology, Punjab Agricultural University, Ludhiana-141001.
2
Department of Soil Sciences, Punjab Agricultural University, Ludhiana-141001.
[Corresponding author E-mail*1: drpkatyal@pau.edu]

Received: 14-07-2015 Accepted: 05-11-2015

A total of twenty one morphologically distinct bacterial strains were isolated from selected-28 samples from sewage treatment
plants (STPs) and one untreated sample from common effluent treatment plant located in Ludhiana district. Tolerance of the
isolates against six heavy metals: Ni, Hg, As, W, Cr and Pb were tested. The isolated strains were characterised phenotypically.
Samples procured from CETP (common effluent treatment plant) was also tested for the elemental profile by using Inductively
coupled Argon Plasma-Atomic Emission Spectroscopy (ICAP-AES) and data revealed a very high level of Cu (52.91 ppm) and Ni (152
ppm). Detailed analysis of bacterial isolates in heavy metals contaminated sites revealed the presence of a variety of pigmented
strains. Morphological characterization of the isolates have shown the prevalence of a number of Gram -ve strains which after
biochemical characterization has been found to belong to genera Proteus, Vibrio, Klebsiella and Enterobacter as revealed by their
biochemical analysis.

Over the years, with the active spread and development of the
industries, Heavy Metals have become ubiquitous and
persistent environmental pollutants. India too is not exempted
from such devastating environmental degradation caused by
these pollutants. Some of these elements are required by
organisms at low concentrations, but their accumulation in
the environment is a serious concern for animal and human
health1. Heavy metals are considered to be more persistent
and stable than organic contaminants such as pesticides or
petroleum by-products and are non-biodegradable2-3.
Their presence in soils may be from natural or anthropogenic
origins4-5. These metals can become mobile in soils depending
on soil pH and their speciation and finally a fraction of the total
mass can leach to aquifer or can become bioavailable to living
organisms6. Discharge of various untreated/undertreated
industrial effluents into water-bodies lead to their build-up in
water. Additionally, heavy metals can accumulate in biological
systems and ultimately be introduced into food web via different
mechanisms7. It is well known that heavy metals can be
extremely toxic as they damage nerves, liver and bones, and
also block functional groups of vital enzymes8-9. Some of the
metals like Ni are also listed as a possible human carcinogen
(group-2B) and associated with reproductive problems and birth
defects10. Metal remediation through common physico-
NAAS Rating (2016)-4.20
chemical techniques is expensive and unsuitable in case of
voluminous effluents containing complexing organic matter and
low metal contamination11.
Microorganisms possess a variety of mechanisms to deal with
high concentrations of heavy metals and often are specific to
one or a few metals12. Microbes have developed mechanisms
to tolerate the metals either by presence of heavy metals
removal through efflux, complexation, or reduction of metal
ions or to use them as terminal electron acceptors in anaerobic
respiration13. Though the applications of genetically engineered
microorganisms (GEMs) in bioremediation have received a
great deal of attention, As GEMs have higher degradative
capacity and have been demonstrated successfully for the
degradation of various pollutants under defined conditions.
However, ecological and environmental concerns and regulatory
constraints are major obstacles for testing GEMs14.
Studies on bacterial diversity in heavy metal contaminated
sites have demonstrated a high diversity of microorganisms15.
These indigenous organisms have not only adapted to the
new environments but have also flourished under them16-17. So
exploiting these organisms can be advantageous over the
introduction of some foreign/alien microbial culture as the
survival of the newly introduced bacterial species under the
conditions existing at contaminated sites may be doubtful while

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 PREVALENCE OF HEAVY METAL RESISTANT BACTERIA (534)

intrinsic flora or resident microbes can be far well acclimatized K2Cr2O7 and Pb (NO3)2 were prepared in sterile deionized water.
and can have better survival rate and faster growth. Application The isolates were subsequently transferred to the increasing
of a judicious consortium of growing metal-resistant cells can concentration. Based on the evaluation, minimum inhibitory
ensure better removal through a combination of bioprecipitation, concentration (MIC) was determined at 30oC after an incubation
biosorption and continuous metabolic uptake of metals after period of 5 days.
physical adsorption. Morphological characterization of Isolates: After the
isolation and purification of the isolates, these were subcultured
Recent studies have shown that the strains (bacteria, yeast
on different defined media including TCBS (Thiosulfate Citrate
and fungi) isolated from contaminated sites possesses
Bile Sucrose) agar, XLD (Xylose lysine deoxycholate) agar,
excellent capability of metal scavenging. Some bacterial strains
Hi-Chrome UTI (Urinary Tract Infection) agar and Salmonella-
possess high tolerance to various metals and may be potential
Shigella (SS) agar, to observe their colony characteristics, on
candidates for their simultaneous removal from wastes.
the basis of which they can be assigned to a particular genera.
Evidently, the stage has already been set for the application of
Biochemical Characterization of the Isolates: Selected
metal-resistant growing microbial cells for metal cleanup. This
isolates were characterized biochemically by using
work mainly focuses on the isolation of tolerant bacterial strains
HiAssorted TM Biochemical test kit KB002 of HiMedia
for their applicability in comprehensive treatment of metal-rich
consisting of a combination of 12 tests (Citrate utilization,
effluents. The use of resident microbes of sewage water for
Lysine utilization, Ornithine utilization, urease detection,
heavy metal clean up will be beneficial as these bacterial
phenylalanine deamination, nitrate reduction, H2S production,
species will be well acclimatized to the ecology of sewage
and fermentation of Glucose, Adonitol, Lactose, Arabinose,
water. This may add on to our knowledge on microbial diversity
Sorbitol) for identification of Gram-negative rods. Additional
in sewage water and to the existing gene pool of heavy metal
biochemical tests performed for the characterization of the
resistance.
isolates include catalase, oxidase, casein hydrolysis, Indole
MATERIAL AND METHODS production, Methyl-Red test and Voges- Proskauer's Test.
Heavy metals estimation: Concentrations of heavy metals
Study area: Analysed the microbial diversity of sewage water
in the untreated sample of CETP were measured by using
samples from 28 different regions of Punjab and untreated
Inductively Coupled Argon Plasma - Atomic Emission
sample from CETP located at Focal point-Ludhiana, from where
Spectroscopy (ICAP-AES).
effluent samples in polypropylene bottles were collected.
Processing of the samples for the isolation of tolerant strains
RESULTS AND DISCUSSION
was carried out followed by their morphological and biochemical
characterization. Inoculation of samples on media supplemented with 0.1mM of
different heavy metals led to the isolation of a total of 40 bacterial
Microbial isolates and maintenance: Isolation of indigenous
isolates, out of which only 21 were found to be morphologically
metal resistant bacteria was carried out using standard
distinct from each other (Table-1). No bacterial growth was
microbiological techniques by which Luria Bertani Agar plates
observed on streaking the sample on media supplemented
supplemented with 0.1 mM concentration of six different heavy
metals i.e. Ni, Hg, As, W, Cr and Pb were used.
Table: Composition of Luria Bertani Agar medium
In vitro metal toxicity analysis to select tolerant strains:
Maximum resistance of the 21 isolates against increasing
concentrations of Ni, Hg, As, W, Cr and Pb on LB agar plates
was evaluated until the strains were unable to grow on the
heavy metal supplemented agar plates. For all the metals,
initial concentration of 0.1 mM was prepared from 1 M stock
solution. The stock solutions of NiCl2, HgCl2, Na2As2O3, NaW,

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 (535) KATYAL, SAHOTA, SINGH AND SIKKA

Tabel-1. Morphological description of bacterial isolates on heavy metal supplemented media.

Sample Hg Cr Ni Pb W
No.
1 + +, dirty white + + +, mango, lemon + + + + +, orange, peach + + + + +, Peach, + + + + +,
yellow, orange turmeric yellow
2 + + +, lemon + + +, lemon + + +, dirty white + + +, Mango + + +. Yellow
3 +, cream + + +, peach + +,dirty whity + + +, creamish + + + +, cream
4 + + + + +, cream + + + +, brown + + +, white, lemon + + +, Creamish + + + +, Dirty
white
5 + + +, peach ++ ++ ++ + + + +, mucoid, cream + + +, Peach + + + + + , Dirty
white
6 +, sindoori + + + +, turmeric + + + +, orange, turmeric + + + +, orange, + + + +, Mango,
peach yellow
7 + + ++ +, no discoloration + +, with brownish + +, with brownish + +, with brownish + +++ +,
discoloration discoloration discoloration brown
8 + +, peach + +, with brownish + +, with brownish + +, with brownish + + +, brown
discoloration discoloration discoloration
9 Ng + + +, brown, dirty white + + +, brown, dirty white + + + + + +, + + + + +, cream
brown, white
10 Ng + + +, brown, dirty white + + +, brown, dirty white + + + + + +, + + + + +, cream
brown, white
11 Ng + + +, brownish + + +, brownish Brownish + + +, brownish
discoloration discoloration discoloration of discoloration
media
12 + + + +, cream + + +, brownish + + +, brownish Brownish + + +, brownish
discoloration discoloration discoloration of discoloration
media
13 Ng Ng Ng No growth Ng
14 Ng +, mucoid, dirty brown Ng Brown mucoid Single mucoid
Me
15 Ng +, mucoid + +, turmeric No growth Ng
16 + + + +, cream Ng + +, cream, mucoid + + +, Peach
17 Ng +, mucoid + +, orange, cream Ng
18 Ng + + +, Peach, mango ++ + + +, Peach, + + +, cream
yellow
19 Ng + + +, mango Ng + + + +, peach, + + + +, mango
white
20 Ng + +, mucoid + +, peach, white + + +, creamish Ng
21 +, cream +++, brownish ++++, turmeric yellow + + , Dirty white ++, mucoid dirty

discoloration white

22 Ng + +, cream, mango + + +, orange cream + + , Dirty white + +, mucoid dirty

white
23 + +, orange + + +, peach, lemon + +, orange cream + + +, orange + +, orange
24 + orange + + + +, lemon, cream + +, cream + + , cream, brown + +, cream,
brown
25 + orange with shine + + +, white, brown + + +, lemon + + , white ++ +
26 Ng + +, cream, brown + +, dirty white + + + , dirty white + + + +, lemon

with Arsenic (As). Most of the bacterial isolates were found to including - XLD, TCBS, UTI and SS agar.
be pigmented but their pigmentation pattern is independent of
Whereby, isolate HM-12 was most likely to belong to Proteus
concentration of heavy metal (Table-2). After obtaining pure
or Vibrio group. HM-3, 5, 11, 16, 18 belong to Enterobacter or
culture of these 21 isolates, their m orphological
Proteus. HM-16 resemble more to Enterobacter aerogenes.
characterization was carried out on the basis of Gram staining
HM-1 and 2 resemble Klebsiella. HM-13 showed the
(Table-3) and colony characteristics on different selected media
characteristic colonies of Pseudomonas. While HM-4 is

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 PREVALENCE OF HEAVY METAL RESISTANT BACTERIA (536)

Table-2. Morphologically distinct isolates selected for Table-3. Specific colony characteristics of the selected
metal tolerance isolates on Differential media.
Isolate No. Proposed Genera Colony Characteristic
12, 4 Proteus, Vibrio harveyi/ fischeri Bluish green colonies with metallic sheen on TCBS
5, 11 Enterobacter, Proteus Grey with black center on XLD
16 Enterobacter aerogens Yellow on XLD
1, 2 Klebsiella Pinkish to purple, mucoid
3, 18 Enterobacter Dark blue colonies on UTI
13 Pseudomonas/Aeromonas Dry, irregular colonies, turquoise on TCBS
4 Shigella Light pink on SS Agar

Table-4. Minimum inhibitory concentration of the heavy

metal against selected bacterial isolates.

(8.087) was quite low. Depending upon elemental profile the

microbial diversity of bacteria resistant to Ni, Cu and Cd was
expected to be high. In another study, some workers isolated
53 different bacterial species from the sediment samples
collected from Krishna Godavari basin, Of these isolates,
79.24% were found to be resistant to 350ppm of Mercury
characterized as Shigella. In a recent study, Bacteria capable
(11.53%), 250ppm of Cadmium (3.77%), 700ppm of Chromate
of accumulating heavy metals were isolated from soil samples
(50.94%) and 250ppm of Zinc (13.20%)19. Similarly, 13 heavy
of Mauritius and identified by standard biochemical test. Out
metal resistant bacteria were isolated from the wastewater of
of the 113 isolates, twelve were capable of growing in the
wadi El Harrach in the east of Algiers and were characterized.
presence of mercury, lead, silver, zinc and copper at varying
These include zinc-, lead-, chromium- and cadmium-resistant
concentrations (1.0-5.0mM) and were identified to belong to
bacteria. The metal-resistant isolates characterized include
the Bacillales. This was confirmed by the sequenced 16S rDNA
both Gram-negative (77%) and Gram-positive (23%) bacteria20.
genes (  491 bp) of all isolates. Two isolates showed 99%
The resistance could have been due to the selective pressure
similarity with Bacillus cereus, four showed 98% similarity
exerted on the organisms by pollution of the marine
with Bacillus subtilis subsp. subtilis while the rest showed
atmosphere with heavy metals.
91% similarity with Bacillus subtilis group when identified in
the GenBank18. Earlier also, Some researchers investigated the mid stream
water quality of Ganga river21. The data revealed that the mid-
Heavy Metal Profile of untreated CETP sample: ICAP-
stream water of river Ganga at Varanasi is invariably
AES analysis of the effluent sample revealed the presence of
contaminated by heavy metals. Although the concentrations
a very high level of contamination by Ni (152ppm) and Cu
of Zn, Cu and Cr remained below the permissible limits of
(52.91ppm), though the level of Cd (0.123), As (0.080) and Pb

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 (537) KATYAL, SAHOTA, SINGH AND SIKKA
Table-5. Biochemical Characterization of the heavy metal tolerant isolates.
Indian standards for drinking water, levels of Cd, Ni and Pb at
three stations, were above the internationally recommended
(WHO) maximum admissible concentrations (MAC). Some
workers reported that concentration of most of the toxic metals
like Al, As, Cd, Cr, Hg, Ni, Pb, Sr and Mn for the year 2010-11
were higher than 2009-10 by a factor of 1.0 to 2.5 μ g/g in
Vasai Creek of Mumbai22. Some earlier workers reported the
heavy metal concentration, in irrigated water from industrial
area of Niani, Allahabad23. The value of Zn, Cu, Pb and Ni was
lower than maximum residue limit proposed by FAO/WHO
with the exception of Cd and Fe which exhibited elevated level.
Heavy metal tolerance of the isolates: The Minimum
Inhibitory Concentration (MIC) of wastewater isolates against
the four heavy metals was determined in solid media by Yamina
et al and ranged from 100 to 1,500 μ g/ml20. A study carried
out earlier revealed that despite the ability of metal tolerant
strains to survive and grow in the presence of Cr, Ag, Hg, the
interactions with these metals may result in metabolic or
physiological changes in these tolerant bacteria24.
Out of the 21 isolates, only five isolates viz. HM-2, HM-3, HM-
5, HM-12 and HM-16 were found to be able to grow in media
supplemented with increasing concentration of heavy metals.
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All the isolates were more tolerant to Pb and W as compare
to Hg, Cr and Ni. Most of the isolates can tolerate upto 4-8
Mm of Pb and W supplemented in LB media. Among the
selected five isolates, HM-2 was found to be least tolerant. It
can tolerate only upto 0.2mM of Hg and 0.4 mM of Cr. Whereas,
maximum tolerance was observed in HM-3 and HM-16 that
can tolerate Hg (1.2-1.4 Mm), Cr (1.0 mM) and Ni (0.8-1.0
mM). HM-12 was moderately tolerant to the selected heavy
metals. A detailed biochemical analysis using the kit as well
as individual tests performed for the characterization of isolates
revealed that the isolates showing high tolerance to heavy
metals (HM-3 and HM-16) were belonging to genera
Enterobacter. The phenotypic characteristics of HM-16 were
very much similar to E. aerogenes. The least tolerant isolate
(HM-2) was found to belong to genera Klebsiella. HM-5 and
HM-12 showed the characteristics similar to genera Proteus
and Vibrio, respectively.
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