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Characterization of Metal Tolerant Serratia spp. Isolates from Sediments of

Uranium Ore Deposit of Domiasiat in Northeast India

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DOI: 10.1007/s40011-013-0236-0

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Characterization of Metal Tolerant Serratia
spp. Isolates from Sediments of Uranium
Ore Deposit of Domiasiat in Northeast
India

Barnali Sarma, Celin Acharya &

S. R. Joshi

Proceedings of the National

Academy of Sciences, India Section B:
Biological Sciences

ISSN 0369-8211

Proc. Natl. Acad. Sci., India, Sect. B Biol.

Sci.
DOI 10.1007/s40011-013-0236-0

1 23
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 Author's personal copy
Proc. Natl. Acad. Sci., India, Sect. B Biol. Sci.
DOI 10.1007/s40011-013-0236-0
RESEARCH ARTICLE
Characterization of Metal Tolerant Serratia spp. Isolates
from Sediments of Uranium Ore Deposit of Domiasiat
in Northeast India
Barnali Sarma • Celin Acharya • S. R. Joshi
Received: 20 December 2012 / Revised: 28 June 2013 / Accepted: 14 August 2013
Ó The National Academy of Sciences, India 2013
Abstract Three metal tolerant Serratia spp. isolates from
sediments of pre-mined uranium ore deposit of Domiasiat,
Meghalaya, India were characterized using morphological,
biochemical and molecular methods. 16S rRNA gene
analysis of the isolates identified one of the isolates (DB-10)
as Serratia nematodiphila with 99.33 % pairwise similarity
whereas remaining two isolates (DB-11 and DB-15) showed
similarity with Serratia marcescens subsp. sakuensis with
similarity of 98.78 and 99.54 % respectively. Randomly
amplified polymorphic DNA (RAPD) and enterobacterial
repetitive intergenic consensus sequence-based PCR (ERIC-
PCR) studies of the isolates revealed genetic diversity
among them. The isolates were found to tolerate all the
five tested metals namely, uranium, cadmium, copper,
zinc and lead and also could resist nine commonly used
antibiotics namely, ampicillin, kanamycin, chloramphen-
icol, erythromycin, gentamicin, aztreonam, tetracycline,
imipenem and ciprofloxin. All the three isolates were
found to be superior in tolerance and resistance to the
reference strain S. marcescens ATCC13880. The isolates
showed high efficiency of uranium (VI) removal ranging
from 90 to 92 % (21.4–21.9 mg/L) and from 65 to 70 %
(309.4–333.2 mg/L) when challenged with 100 lM
Electronic supplementary material The online version of this
article (doi:10.1007/s40011-013-0236-0) contains supplementary
material, which is available to authorized users.
B. Sarma  S. R. Joshi (&)
Microbiology Laboratory, Department of Biotechnology and
Bioinformatics, North-Eastern Hill University, Umshing,
Shillong 793022, Meghalaya, India
e-mail: srjoshi2006@yahoo.co.in
C. Acharya
Molecular Biology Division, Bhabha Atomic Research Centre,
Trombay, Mumbai 400085, Maharashtra, India
(23.8 mg/L) and 2 mM (476 mg/L) uranyl nitrate solu-
tions respectively within 1 h of incubation.
Keywords Serratia isolates  Uranium deposit 
Metals  Antibiotics  Genetic diversity
Introduction
Domiasiat (25°200 N, 91°130 E; Survey of India Toposheet
78 O/3) in West Khasi hills district of Meghalaya, North-
east India was identified with huge deposits of uranium by
India’s Atomic Mineral Division in 1984 and it has been
characterized as the largest, richest and near-surface
sandstone-type uranium deposits in India containing 9.22
million tonnes of ore reserves, with an average ore grade of
0.1 % U3O8 [1, 2].
Microbial populations from metal rich sites are known
to show higher resistance to heavy metals as compared to
populations of non-contaminated or non-metal rich sites
[3–8]. Several microorganisms are known to undergo
adaptation to such environments through different mecha-
nisms like, development of permeability barriers, intra- and
extra-cellular sequestration, efflux pumps, enzymatic
detoxification, reduction, biosorption, bioprecipitation,
transport mechanisms and chelation, [6, 8–10] thereby
reducing the cellular toxicity of metallic contaminants.
Alonso et al. [11] implicated a cluster of genes to be
involved in antibiotic and heavy-metal resistance of a
clinical isolate of gram-negative bacterium, Stenotropho-
monas (Xanthomonas) maltophilia. Metal and antibiotic
resistance sometimes get transferred together among
microorganisms in the environment imparting the organism
with dual tolerance [6, 12]. Such resistance mechanisms
and adaptations are the basis for the use of microorganisms
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 Author's personal copy
in bioremediation approaches [9]. Wild isolates of bacteria
exhibiting multi-metal and actinide tolerance, acid toler-
ance and aerobic metabolism are reported as promising
agents for in situ bioremediation of actinide and metal-
contaminated areas [13]. Although, there is a study on the
biosorption of uranium by Serratia marcescens from the
present site correlated to U uptake behaviour [7], but the
multi-metal and antibiotic resistance strategies for
homeostatic behaviour in Serratia nematodiphila and S.
marcescens subsp. sakuensis have not been reported from
the studied site.
The present investigation was aimed to isolate and
characterize uranium-tolerant Serratia species from the
pre-mined uranium ore bearing site and their comparison
with a type strain, S. marcescens ATCC13880. Molecular
diversity among these isolates along with the type strain
was studied using randomly amplified polymorphic DNA
(RAPD) and enterobacterial repetitive intergenic consensus
sequence-based PCR (ERIC-PCR) techniques. Minimum
inhibitory concentration (MIC) of metals including ura-
nium (U), antibiotic resistance, uranyl biosorption potential
and evaluation of their viability at toxic concentrations of
U were used to find functional diversity among them.
Material and Methods
Sample Collection, Analysis and Isolation of the Test
Isolates
Sediment samples from water bodies were collected from
three different locations of the proposed uranium rich mining
site of Domiasiat (25° 200 N 91° 130 E; Survey of India To-
posheet 78 O/3) area of Meghalaya in Northeast India. Sedi-
ments from the sites were collected and brought to the
laboratory in sterile condition at 4 °C. pH of all samples was
measured using pH meter (Systronics, India). The trace metals
like copper (Cu) cadmium (Cd), zinc (Zn) and lead (Pb) were
analysed using atomic absorption spectrophotometer (AAS,
Perkin Elmer 3110), while U was estimated using inductively
coupled plasma mass spectrometer (ICP-MS, Perkin Elmer
Elan DRC) from the acid digested samples.
For the isolation of metal tolerant bacteria, 10 grams of
each sample was inoculated in 100 ml of low phosphate
broth [Tris 14.5 g, NaCl 4.68 g, KCl 1.5 g, NH4Cl 1.0 g,
glycerol 5.0 ml, Na2SO4 0.043 g, CaCl2 0.03 g] at pH 7.5
in Erlenmeyer flasks enriched with 1 mM uranium as UO2
(NO3)2. 6H2O and incubated at 30 °C at 150 rpm for 24 h.
Serial ten-fold dilutions of the enrichment cultures were
inoculated onto tryptone soy agar (HiMedia, India), diluted
to 0.5 % in triplicates, enriched with 2 mM of U [UO2
(NO3)26H2O], 2 mM Cu [CuSO45H2O], 0.5 mM of Cd
[Cd (NO3)25H2O], 1 mM of Zn [ZnSO47H2O] and
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B. Sarma et al.
0.5 mM Pb [Pb(NO3)2] and incubated at 32 °C for
24–72 h. Colonies were randomly picked up from the
metal supplemented plates. Purity of the culture was con-
firmed by sub-culturing on nutrient agar plates and repe-
ated for three times. Confirmed pure cultures were
preserved using 15 % glycerol stocks for further use.
Reference Culture
A reference strain S. marcescens subsp. marcescens Bizio
1823 ATCC13880 (S. marcescens ATCC13880) was pro-
cured from American type Culture Collection (ATCC),
USA and used for comparative studies along with the
natural isolates from the sediment samples collected from
the study site as well as the natural isolates available in the
laboratory of the authors [7].
Identification of the Isolates by 16S rRNA Gene
Analysis
Genomic DNA was extracted from the bacterial isolates
using yeast and bacterial genomic DNA kit (HiMedia,
India). 16S rRNA gene sequences were amplified as
described earlier [8]. DNA amplification was carried out in
Gene AMP PCR system 9700 (Applied Biosystems, USA)
with an initial denaturation step of 94 °C for 5 min, fol-
lowed by 30 cycles consisting of denaturation at 94 °C for
1 min, annealing at 55 °C for 1 min, and extension at
72 °C for 2 min, followed by a final extension step of
72 °C for 5 min. Approximately l,500 nucleotides were
amplified using PCR. Amplified products were sequenced
bi-directionally with Big Dye (3.1) terminator protocol.
The phylogenetic neighbors of the isolates were obtained
using the Basic local alignment search tool (BLAST)
program [14] against EzTaxon server version 2.1 [15].
Molecular Evolutionary Genetics Analysis software
(MEGA version 4) was used for phylogenetic analyses
[16]. The sequences of identified phylogenetic neighbor
were aligned with the sequences of test isolates using
Clustal W inbuilt with MEGA 4. Deinococcus radiodurans
M20539T was used as the outgroup organism. Neighbor-
joining method was employed to construct the phyloge-
netic tree with 1,000 bootstrap replications to assess nodal
support in the tree. The partial 16S rRNA gene sequences
of the isolates were submitted to NCBI GenBank and
accession numbers obtained.
Diversity Analysis Using Molecular Methods
RAPD Banding Patterns
Ten random primers RBa1 (50 -AATCGCGCTG-30 ), RBa2
(50 -AATCGGGCTG-30 ), RBa3 (50 -ACACACGCTG-30 ),
 Author's personal copy
Serratia spp. Isolates from Sediments of Uranium Ore Deposit
RBa4 (50 -ACAGGGGTGT-30 ), RBa5 (50 -ACCACCCACC-
30 ), RBa6 (50 -ACTTCGCCAC-30 ), RBa7 (50 -AGCCTGAG
CC-30 ), RBa8 (50 -AGGCGGCAAG-30 ), RBa9 (50 -AGGT
GACCGT-30 ) and RBa10 (50 -ATCCTGCCTG-30 ), procured
as Bacterial RAPD primers kit (Merck, India) were used for
RAPD study. Amplification was done according to the man-
ufacturer’s instruction. The amplified products were separated
on 2 % agarose gel prepared with TBE buffer at 80 V and
visualized using ethidium bromide staining. The size of the
amplified fragments was determined using 100 bp and 1 kb
DNA ladders (Merck, India) as molecular weight size mark-
ers. The analysis was repeated thrice to obtain consistent and
reproducible bands. Four primers were found to give consis-
tent results. Each band was treated as a separate character and
scored as present (1) or absent (0) to obtain a combined binary
data matrix. A dendrogram was constructed using unweighed
pair group method and arithmetic mean (UPGMA) clustering
method in NTSYSpc software.
ERIC-PCR Banding Patterns
Enterobacterial repetitive intergenic consensus sequence-
based PCR (ERIC-PCR) was performed to detect differ-
ences in number and distributions of these bacterial
repetitive sequences in the bacterial genome. ERIC-PCR
was carried out according to Versalovic et al. [17] using a
primer pair ERIC 1R 50 -ATG TAA GCT CCT GGG GAT
TCA C-30 and ERIC 2 50 -AAG TAA GTG ACT GGG GTG
AGC G-30 . The amplified products were separated and a
dendrogram was constructed as done for RAPD analysis.
Morphological and Biochemical Characterization
of the Isolates
The selected S. marcescens isolates and the type strain
ATCC13880 were characterized morphologically and bio-
chemically. Morphological characteristics of the isolates
and the type strain were evaluated after gram staining.
Different biochemical properties were determined using
different tests like growth under anaerobic conditions,
production of catalase, oxidase, urease, gelatinase, lipase,
DNase and utilization of different carbohydrates like dex-
trose, sucrose, lactose, mannose, maltose, galactose, glyc-
erol, D-arabinose and D-cellobiose.
Determination of Minimal Inhibitory Concentration
(MIC) of Metals
Analytical grade salts of uranyl nitrate [UO2(NO3)2.
6H2O], copper sulphate [CuSO45H2O], cadmium nitrate
[Cd (NO3)25H2O], zinc sulphate [ZnSO47H2O] and lead
nitrate [Pb (NO3)2] were used to prepare stock solutions.
The stock solutions of these metals were filter sterilized
through a 0.22 lm nitrocellulose membrane filter (Milli-
pore, India). The selected isolates of present study and the
type strain S. marcescens ATCC13880 were grown to mid-
exponential phase in low phosphate broth (LPB) and tested
their tolerance to increasing concentration of heavy metals
on Low Phosphate Agar (LPA) according to Sarma et al.
[8]. Concentration of metals was increased as factor of 0.5,
starting from 0.5 mM to get the MIC value. The minimum
concentration of metal that completely prevented growth
on agar plates after 72 h incubation at 32 °C was taken to
be the MIC.
Determination of Antibiotic Sensitivity of the Isolates
The characterized isolates and the reference strain were
screened for antibiotic resistance on MHA plates against 9
commonly used antibiotics viz.: ampicillin (10 lg)—A,
kanamycin (30 lg)—K, chloramphenicol (10 lg)—C,
erythromycin (10 lg)—E, gentamicin (10 lg)—G, aztreo-
nam (30 lg)—Ao, tetracycline (10 lg)—T, imipenem
(10 lg)—I and ciprofloxin (5 lg)—Cf (HiMedia, India). The
results were recorded as resistant (R), intermediate (I) and
susceptible (S) on the basis of the diameter of the inhibition
zone from the zone size interpretative chart supplied by the
manufacturer.
Uranyl Biosorption Capacity of the Isolates
Uranyl biosorption study was done according to Sarma
et al. [8]. Mid-exponential phase cells of all the isolates and
the reference strain was used for the study. Four different
concentrations of uranyl nitrate solutions viz., 100 lM,
500 lM, 1 mM, and 2 mM were taken. U binding poten-
tials were then estimated by using the arsenazo III method
[18]. Each treatment consisted of 3 replicates.
The viability test to evaluate the tolerance of strains to
acidic condition (pH 3.5) and uranium (100 lM and 2 mM)
were done as described Kumar et al. [7] and Sarma et al. [8].
Results and Discussion
Biogeochemical Properties of the Samples
and Selection of Isolates
Metal enrichment based method was employed to isolate
bacteria from the samples which yielded 54 bacterial iso-
lates. U concentration was found highest in Kylleng mining
site 1 (Table 1). Other metals were within the range of
background concentration of trace elements as estimated in
non-anthropogenic soils [19]. Molecular characterization
with 16S rRNA gene sequences revealed that 24 (44 %) of
these isolates were Serratia sp. ([98 % maximum identity)
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B. Sarma et al.

Table 1 Location and biogeochemical properties of the sampling sites

Sampling sites Geographic coordinates pH Conc. of metals (in ppm) Totala Serratia spp. isolates (%)
North East U Cu Zn Cd Pb

Kylleng mining site 1 25°190 14.800 91°120 46.600 5.6 480 28.8 377.0 0.9 23 22 10 (45.45)
Kylleng mining Site 2 25°190 09.100 91°120 52.300 5.3 100 7.3 115.2 2.7 11 11 5 (45.45)
Phudsyngkai mining site 25°190 06.500 91°120 52.800 5.35 200 16 165.3 2.7 22 21 9 (42.86)
a
Total isolates for bacteria including Serratia

when the BLAST program was performed against the
nucleotide database of NCBI. The remaining 30 isolates
(56 %) comprised of 11 different genera, viz., Pseudo-
monas, Brevundimonas, Comamonas, Achromobacter,
Stenotrophomonas, Ochrobactrum, Paenochrobactrum,
Alcaligenes, Enterobacter, Bacillus and Acinetobacter.
Based on dominance and superior metal tolerance ability,
three representative isolates of Serratia (DB-10, DB-11
and DB-15) were selected for further study.
Identification by 16S rRNA Gene Sequencing
BLAST results of 16S rRNA gene sequence of the isolate
DB-10 showed 99.339 % pairwise similarity with S. nem-
atodiphila whereas DB-11 and DB-15 showed similarity
with S. marcescens subsp. sakuensis by 98.785 and
99.54 % respectively in EzTaxon database server [15]
(Fig. 1). GenBank accession numbers for the sequences
obtained are HQ696505, HQ696506 and JQ074042 for
DB-10, DB-11 and DB-15 respectively.
Diversity Analysis Using Molecular Methods
Out of ten RAPD primers, four primers, namely RBa1,
RBa2, RBa3 and RBa7 produced consistent and repro-
ducible banding patterns. These four primers produced a
total of 25 amplified fragment bands, with an average 6.25
bands per primer. Of these, 9 were polymorphic and 16
were monomorphic (SFig. 1a–d). A dendrogram based
on the combined similarity matrix was generated using
UPGMA (NTSYSpc software) with pair-wise coefficient
similarity based on presence (1) and absence (0) of bands
with the 4 RAPD primers. The dendrogram revealed that
the 3 test isolates and the type strain S. marcescens
ATCC13880 were different from each other. The highest
similarity was observed between DB-11 and ATCC13880
with 0.68 similarity coefficient. The similarity coefficients
ranged from 0.4 to 0.68 (Fig. 2).
ERIC-PCR fingerprinting revealed similar genetic pat-
terns as RAPD. The band size ranged from *220 bp to
*2,300 bp (SFig. 2). The dendrogram revealed two major
clusters with similarity coefficient 0.30. The isolate DB-15
was found to be in one cluster while the other cluster had
two isolates and the type strain. However, DB-11 and the
type strain S. marcescens ATCC13880 had the highest
similarity coefficient of 0.80 (Fig. 3).
Morphological and Biochemical Characterization
of the Isolates
All of three selected isolates along with the type strain S.
marcescens ATCC13880 were found to be gram-negative
rods, motile, fermentative, facultatively anaerobic, oxidase-
negative and catalase-positive. They produced gelatinase,
lipase, nitrate reductase, and DNase. They showed positive
results for citrate utilisation by Voges-Proskauer test while
negative results for methyl red test, indole production, hydro-
gen sulphide production, urease test and amylase test. All the
isolates, including the type strain, produced acid from man-
nose, maltose, galactose, and glycerol and none of them pro-
duced acid from lactose, sucrose, D-cellobiose and D-arabinose.
Dextrose could not be utilised by DB-10 and DB-11.

Fig. 1 Neighbor-joining tree

based on 16S rRNA gene
sequences depicting the
phylogenetic relationships
between the isolates and the
related species with
Deinococcus radiodurans
M20539T taken as the out-
group organism (DB-15, DB-11
and DB-10 are characterized
isolates)

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Serratia spp. Isolates from Sediments of Uranium Ore Deposit

Fig. 2 Combined dendrogram

constructed from banding
pattern of the four RAPD
primers with unweighed pair
group method arithmetic mean
(UPGMA) clustering
programme in NTSYS-pc
software (DB-15, DB-11 and
DB-10 are characterized
isolates; ATCC is S. marcescens
ATCC13880)

Fig. 3 Dendrogram constructed

from ERIC-PCR banding
patterns with unweighed pair
group method arithmetic mean
(UPGMA) clustering
programme in NTSYSpc
software (DB-15, DB-11 and
DB-10 are characterized
isolates; ATCC is S. marcescens
ATCC13880)

Metal Tolerance and Antibiotic Resistance
of the Isolates
All the isolates tolerated considerably higher concentra-
tions of metals than the type strain (Fig. 4). Patterns of
metal tolerance varied among the isolates. In natural
environments, metals can occur in different availabilities
for bacteria due to the presence of other agents which can
bind with metals [20]. Metal tolerance of bacteria varies
with different media used for tolerance study [8]. Since
metal availability depends on the media components, it can
be assumed that minimal and defined media LPA mini-
mizes the probability of complex formation with metals to
a great extent.
The order of toxicity of the metals to the bacteria was
different. Pb and Cd were found to be highly toxic for all the
four bacterial cultures tested and they shared same MIC
values of these two metals viz., 0.5, 1.5, 2 and 2 mM for
ATCC 13880, DB-10, DB-11 and DB-15 respectively. Cu
was the least toxic metal with MIC values of 4, 5 and 5 mM
for DB-10, DB-11 and DB-15 respectively whereas U and Zn
with MIC value 1.5 mM took that position in case of refer-
ence strain S. marcescens ATCC13880 (Fig. 4).
Two antibiotics G10 and I10 consistently inhibited
bacterial growth. All the isolates including type strain
showed resistance towards A10, K30 and Ao30. In case of
the antibiotics C10, E10 and T10, all the test isolates
showed resistance whereas the reference strain S. marces-
cens ATCC13880 showed intermediate result (Table 2).
The characterised wild isolates were found to tolerate
considerable amount of metals and were found to resist
most of the commonly used antibiotics. Due to the effi-
ciency of natural microorganisms isolated from contami-
nated habitats earlier reports have proposed the utilization
of these bacteria in bioremediation processes following
various approaches such as, biotransformations [21, 22],
mobilization and/or immobilization of the metals [23–27],
biosorption [28, 29], uptake of metals [4, 30], induction of
metal precipitation [31, 32] or alteration of metal specia-
tion caused by microbe mediated redox changes [33].

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B. Sarma et al.

Fig. 4 MIC of five different

metals in milliMolar (mM)
concentrations in LPA

Table 2 Antibiotic sensitivity of the isolates and type strain against commonly used antibiotics
Isolates Sensitivitya to common antibioticsb
Cf5 C10 T10 E10 G10 I10 A10 K30 Ao30

ATCC13880 S I I I S S R R R
DB-10 I R R R S S R R R
DB-11 I R R R S S R R R
DB-15 S R R R S S R R R
a
R Resistant, I Intermediate, S Susceptible
b
Cf5-ciprofloxin (5 lg), C10-chloramphenicol (10 lg), T10-tetracycline (10 lg), E10-erythromycin (10 lg), G10-gentamicin (10 lg), I10-
imipenem (10 lg), A10-ampicillin (10 lg), K30-kanamycin (30 lg), Ao30-aztreonam (30 lg)

Uranyl Biosorption Capacity and Viability
of the Isolates
For further use of the wild Serratia spp. isolates in biore-
mediation approaches, uranyl biosorption potential of the
isolates was evaluated with respect to incubation time
(5 min to 24 h) as well as initial uranium concentrations
(100 lM–2 mM; 23.8–476 mg/L). The isolates showed
high efficiency of uranium (VI) removal ranging from 90 to
92 % (21.4–21.9 mg/L) when challenged with 100 lM
(23.8 mg/L) and from 65 to 70 % (309.4–333.2 mg/L)
when challenged with 2 mM (476 mg/L) uranyl nitrate
solutions at pH 3.5. The reference strain showed 20 %
(4.76 mg/L) removal from 100 lM and 15 % (71.4 mg/L)
uranium removal from 2 mM test solutions at pH 3.5
(Fig. 5) which correlates with earlier results [7]. Uranium
remained stable under the experimental conditions and did
not precipitate as seen in the abiotic controls. None of the
wild isolates and the reference strain could remove ura-
nium from uranyl nitrate solution at pH [7. All the three
isolates remain viable under the stress of acidic condition
(pH 3.5) and toxic concentrations of uranium U(VI)
(100 lM–2 mM). Though the colony forming unit (CFU)
count of the reference strain was lower than that of Dom-
iasiat isolates, it was viable under the test conditions (Data
not shown).
The results of the present study were coherent with the
findings of other investigators indicating the increased
occurrence of metal-tolerant bacteria with the increase of
heavy metal/radionuclide concentration in metal-contami-
nated sites [34–37]. Aiking et al. [38] stated that resistance
or tolerance of toxic metals in bacteria probably reflects the
degree of environmental contamination with these sub-
stances and may be directly related to exposure of bacteria
to them. Factors such as agronomic practices are also
known to influence microflora which relates to immediate
environmental conditions [39]. The phenomenon of resis-
tance and/or tolerance capacities is generally regarded as
adaptation by microbes under adverse conditions like
exposure to metal in contaminated sites [6].

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Serratia spp. Isolates from Sediments of Uranium Ore Deposit
Fig. 5 Uranium binding by the
three wild isolates and reference
strain S. marcescens
ATCC13880. Error bars denote
standard deviation (n = 3)
The reference strain S. marcescens ATCC13880 was
showing less metal and antibiotic resistance than the wild
isolates indicating higher adaptability of the wild isolates to
metal rich conditions. Uranyl biosorption potential of the
reference strain was also very less as compared to the wild
isolates. It can be assumed that resistance capacity in wild
bacteria isolated from uranium rich site in Domiasiat is
more than the same bacteria from other source and can be
utilised for bioremediation approaches.
Conclusion
In the present study, three metal tolerant Serratia spp.
isolates from sediment samples of pre-mined uranium ore
deposits of Meghalaya were characterized and compared
with the reference strain S. marcescens ATCC13880. The
wild isolates were found to be tolerant to higher con-
centrations of metals including U when compared to the
type strain. The Serratia isolates showed significant
resistant to commonly used antibiotics. Apart from being
tolerant to heavy metals and antibiotics, the isolates were
also found to possess good uranyl biosorption potential of
90–92 and 65–70 % when challenged with 100 lM and
2 mM uranyl nitrate solutions respectively. These poten-
tials suggest that the wild Serratia isolates can be
explored for use in bioremediation of multi-metal con-
taminated sites.
Acknowledgments The financial support received from the
Department of Atomic Energy, Bhabha Atomic Research Centre
(BARC-BRNS), Govt. of India is thankfully acknowledged.
References
1. Raju D, Selvam RP, Virnave SN (1989) Characterisation of the
upper cretaceous lower Mahadek Sandstone and its uranium
mineralisation in the Domiasiat-Gomaghat Pdengshakap area,
Meghalaya, India. Expl Res Atom Miner 2:1–27
2. Sengupta B, Bahuguna R, Kumar S, Singh R, Kaul R (1991)
Discovery of a sandstone-type uranium deposit at Domiasiat,
West Khasi hills district, Meghalaya, India. Curr Sci 61:46–47
3. Gadd GM (2000) Bio remedial potential of microbial mecha-
nisms of metal mobilization and immobilization. Curr Opin
Biotechnol 11:271–279
4. Francis A, Gillow J, Dodge C, Harris R, Beveridge T, Papenguth
H (2004) Uranium association with halophilic and non-halophilic
bacteria and archaea. Radiochim Acta 92:48–488
5. Schmidt A, Haferburg G, Sineriz M, Merten D, Buchel G, Kothe
E (2005) Heavy metal resistance mechanisms in actinobacteria
for survival in AMD contaminated soils. Chem Erde Geo Chem
65:131–144
6. Sarma B, Acharya C, Joshi SR (2010) Pseudomonads: a versatile
bacterial group exhibiting dual resistance to metals and antibi-
otics. Afr J Microbiol Res 4(25):2828–2835
7. Kumar R, Acharya C, Joshi SR (2011) Isolation and analyses of
uranium tolerant Serratia marcencens strains and their utilization
for aerobic uranium U(VI) bioadsorption. J Microbiol 49:
568–574. doi:10.1007/s12275-011-0366-0
8. Sarma B, Acharya C, Joshi SR (2012) PGP and metal bioad-
sorption activity of metal tolerant Pseudomonas aeruginosa iso-
late characterized from uranium ore deposit. Proc Natl Acad Sci
India Sect B Biol Sci. doi:10.1007/s40011-012-0136-8
9. Haferburg G, Kothe E (2007) Microbes and metals: interactions
in the environment. J Basic Microbiol 47(6):453–467
10. Achal V, Pan X, Zhang D (2011) Remediation of copper-con-
taminated soil by Kocuria flava CR1, based on microbially
induced calcite precipitation. Ecol Eng 37:1601–1605
11. Alonso A, Sanchez P, Martinez JL (2000) Stenotrophomonas
maltophilia D457R contains a cluster of genes from gram-posi-
tive bacteria involved in antibiotic and heavy-metal resistance.
Microb Agents Chemother 44:1778–1782
123
 Author's personal copy
12. Calomiris JJ, Armstrong JL, Seidler RJ (1984) Association of metal
tolerance with multiple antibiotic resistance of bacteria isolated
from drinking water. Appl Environ Microbiol 47:1238–1242
13. Choudhary S, Sar P (2009) Characterization of a metal resistant
Pseudomonas sp. isolated from uranium mine for its potential in
heavy metal (Ni2?, Co2?, Cu2? and Cd2?) sequestration. Biore-
sour Technol 100:2482–2492
14. Altschul SF, Madden TL, Schaeffer AA, Zhang J, Zhang Z,
Miller W et al (1997) Gapped BLAST and PSI-BLAST: a new
generation of protein database search programs. Nucleic Acids
Res 25:3389–3402
15. Chun J, Lee JH, Jung Y, Kim M, Kim S, Kim BK, Lim YW
(2007) EzTaxon: a web-based tool for the identification of pro-
karyotes based on 16S ribosomal RNA gene sequences. Int J Syst
Evol Microbiol 57:2259–2261
16. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular
evolutionary genetics analysis (MEGA) software version 4.0.
Mol Biol Evol 24:1596–1599
17. Versalovic J, Schneider M, De Brujin FJ, Lupski JR (1994)
Genomic fingerprinting of bacteria using the repetitive sequence
based polymerase chain reaction. Methods Mol Cell Biol 5:25–40
18. Savvin SB (1961) Analytical use of arsenazo III: determination of
thorium, zirconium, uranium and rare earth elements. Talanta
8:673–685
19. Burt R, Wilson MA, Mays MD, Lee CW (2003) Major and trace
elements of selected pedons in the USA. J Environ Qual
32:2109–2121
20. Hassen A, Saidi N, Cherif M, Boudabous A (1998) Resistance of
environmental bacteria to heavy metals. Biores Technol 64:7–15
21. Lovley D (1993) Dissimilatory metal reduction. Ann Rev
Microbiol 47:263–290
22. Beller H (2005) Anaerobic, nitrate-dependent oxidation of U(IV)
oxide minerals by the chemoöithoauthotrophic bacterium Thi-
obacullus denitrificns. Appl Environ Microbiol 71:2170–2174
23. Francis A (1998) Biotransformation of uranium and other actin-
ides in radioactive wastes. J Alloys Compd 271–273:78–84
24. Banaszak J, Rittmann B, Reed D (1999) Subsurface interactions
of actinide species and microorganisms: implications for the
bioremediation of actinide-organic mixtures. J Radioanal Nucl
Chem 241:385–435
25. Nies D (1999) Microbial heavy-metal resistance. Appl Microbiol
Biotechnol 51:730–750
26. Lloyd J, Lovley D (2001) Microbial detoxification of metals and
radionuclides. Curr Opin Biotechnol 12:248–253
27. Achal V, Pan X, Zhang D (2012) Bioremediation of strontium
(Sr) contaminated aquifer quartz sand based on carbonate
123
View publication stats
B. Sarma et al.
precipitation induced by Sr resistant Halomonas sp. Chemosphere
89:764–768
28. Selenska-Pobell S, Panak P, Miteva V, Boudakov I, Bernhard G,
Nitsche H (1999) Selective accumulation of heavy metals by
three indigenous Bacillus strains, B. cereus, B. megatherium and
B. sphaericus, from drain waters of a urnium waste pile. FEMS
Microb Ecol 29:59–67
29. Hafez M, Ibrahim M, Abdel-Razek A, Abu-Shady M (2002)
Biosorption of some ions on different bacterial species from
aqueous and radioactive waste solutions. J Radioanal Nucl Chem
252:179–185
30. Suzuki Y, Banfield JF (2004) Resistance to, and accumulation of,
uranium by bacteria from a uranium contaminated site. Geomi-
crobiol J 21:113–121
31. Renninger N, McMahon K, Knopp R, Nitsche H, Clark D,
Keasling J (2001) Uranyl precipitation by biomass from an
enhanced biological phosphorus removal reactor. Biodegradation
12:401–410
32. Renninger N, Knopp R, Nitsche H, Clark D, Keasling J (2004)
Uranyl precipitation by Pseudomonas aeruginosa via controlled
polyphosphate metabolism. Appl Environ Microbiol 70:7404–7412
33. Bosecker K (1997) Bioleaching: metal solubilization by micro-
organisms. FEMS Microbiol Rev 20:59–64
34. Angle JS, Chaney RL, Rhee D (1993) Bacterial resistance to
heavy metals related to extractable and total metal concentrations
in soil and media. Soil Biol Biochem 25:1465–1466
35. Roane TM, Kellogg ST (1996) Characterization of bacterial
communities in heavy metal contaminated soils. Can J Microbiol
42:593–603
36. Kumar R, Nongkhlaw M, Joshi SR, Acharya C (2013) Uranium
(U)-tolerant bacterial diversity from U ore deposit of Domiasiat in
North-East India and its prospective utilisation in bioremediation.
Microbes Environ 28(1):33–41. doi:10.1264/jsme2.ME12074
37. Nongkhlaw M, Kumar R, Acharya C, Joshi SR (2012) Occur-
rence of horizontal gene transfer of PIB-type ATPase genes
among bacteria isolated from the uranium rich deposit of domi-
asiat in North East India. PLoS One 7(10):e48199. doi:
10.1371/journal.pone.0048199
38. Aiking H, Stinamn A, Van Ganderen C, Van Heerikhuizen H,
Vant Riet J (1984) Inorganic phosphate accumulation and cad-
mium detoxification in Klebsiella aerogenes NCTC 418 growing
in continuous culture. Appl Environ Microbial 47:374–377
39. Kumar D, Shivay YS, Dhar S, Kumar C, Prasad R (2013) Rhi-
zospheric flora and the influence of agronomic practices on
them—a review. Proc Natl Acad Sci India Sect B Biol Sci
83(1):1–14