Journal of Environmental Science and Health, Part A

Toxic/Hazardous Substances and Environmental Engineering

ISSN: 1093-4529 (Print) 1532-4117 (Online) Journal homepage: http://www.tandfonline.com/loi/lesa20

Arsenic accumulating and transforming bacteria

isolated from contaminated soil for potential use
in bioremediation

Suchanda Banerjee , Sudeshna Datta , Dhrubajyoti Chattyopadhyay &

Priyabrata Sarkar

To cite this article: Suchanda Banerjee , Sudeshna Datta , Dhrubajyoti Chattyopadhyay

& Priyabrata Sarkar (2011) Arsenic accumulating and transforming bacteria isolated from
contaminated soil for potential use in bioremediation, Journal of Environmental Science and Health,
Part A, 46:14, 1736-1747, DOI: 10.1080/10934529.2011.623995

To link to this article: https://doi.org/10.1080/10934529.2011.623995

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Journal of Environmental Science and Health Part A (2011) 46, 1736–1747
Copyright
C Taylor & Francis Group, LLC

ISSN: 1093-4529 (Print); 1532-4117 (Online)

DOI: 10.1080/10934529.2011.623995

Arsenic accumulating and transforming bacteria isolated

from contaminated soil for potential use in bioremediation

SUCHANDA BANERJEE1, SUDESHNA DATTA1, DHRUBAJYOTI CHATTYOPADHYAY2

and PRIYABRATA SARKAR1
1
Department of Polymer Science and Technology, University of Calcutta, Kolkata, India
2
Dr. B.C. Guha Centre for Genetic Engineering and Biotechnology, University of Calcutta, Kolkata, India

Arsenic (As) is a metalloid and considered harmful due to its toxic and carcinogenic effects. Removal of arsenic is of great importance
for human welfare. The main objective of this study was to isolate arsenic-resistant bacteria that are capable of removing arsenic from
the environment. Soil samples were collected from an arsenic-affected area of West Bengal, India and 10 different bacterial strains
were isolated. The minimum inhibitory concentration (MIC) values of the isolates varied widely in the range 50–125 mM (As) as
arsenate and 10–100 mM (As) as arsenite. TEM and EDAX analysis were done to confirm intracellular accumulation of arsenic. The
16s RNA and phylogenetic analysis showed that seven isolates belonged to γ -proteobacterium, two isolates belonged to Firmicutes
and one was identified as Kocuria genera. Some of these bacteria could oxidize arsenite to arsenate and all others could reduce
arsenate to arsenite. The growth pattern of the bacterial strains in presence and absence of arsenic was also observed. All the 10
isolates exhibited multiple heavy metal (like Ni, Zn, Cu, Pb, Co, etc.) tolerances. Thus, these new bacterial strains could conveniently
be used for bioremediation of soil and effluents and the enzymes produced by them may be used for commercial exploitation.
Keywords: Minimum inhibitory concentration, intracellular accumulation, arsenate reduction, arsenite oxidation.

Introduction linked to cancer of the bladder, lungs, skin, kidney, nasal

Heavy metal pollution has become one of the most severe
threats to environment and human health.[1] Most of the
heavy metals have toxic effects on living organisms when
exceeding a threshold level.[2] Arsenic is a toxic metalloid
and is widely distributed in the environment due to natural
geochemical processes and anthropogenic activities.[3] The
most common oxidation states of arsenic in the environ-
ment are the pentavalent arsenate As[V] and the trivalent
arsenite As[III]. Of these two forms, As[III] is much more
toxic than As[V].[4] Arsenite can bind to sulfhydryl groups
and dithiols groups of proteins, whereas arsenate can act
as a chemical analog of phosphate and can inhibit oxida-
tive phosphorylation.[5] The scientific literature shows that
arsenic is one of the heavy metals that has been recognized
as a potent human toxin with reports of many diseases,
such as thickening and discoloration of the skin, stomach
pain, nausea, vomiting; diarrhoea; numbness in hands and
feet; partial paralysis; and blindness. Arsenic has also been
Address correspondence to Priyabrata Sarkar, Department
of Polymer Science and Technology, University of Cal-
cutta, 92 A.P.C. Road, Kolkata 700009, India; E-mail:
sarkarpriya@gmail.com
Received April 1, 2011.
passages, liver, and prostate.
Due to toxic and carcinogenic effects of arsenic, World
Health Organization (WHO) has set a permissible limit of
0.01 mg/L for arsenic in drinking water, which has been
adopted by most of the industrial countries. Despite this,
national standards for arsenic in most Asian countries re-
mains at 0.05 mg/L, due to economic considerations and
the lack of tools and techniques to measure accurately at
lower concentrations. Presently, the extent of groundwa-
ter arsenic contamination in West Bengal has reached an
alarming situation as nine districts of West Bengal have
been reported to have groundwater arsenic concentrations
above 0.05 mg/L and several people have been affected by
arsenic poisoning.[6]
Thus, removal of arsenic is of great importance for hu-
man welfare. The conventional techniques such as chem-
ical precipitation, chemical oxidation and reduction, ion
exchange, filtration, reverse osmosis, etc. are used for re-
moving heavy metal ions from dilute solutions. Further-
more, these techniques of removal are often inappropriate
or expensive, particularly when unwanted heavy metals are
present in very low concentration or in large solution vol-
ume.[7] In recent years, bioremediation of heavy metals us-
ing microorganisms has received a great deal of attention,
not only as a scientific novelty but also for its potential.[8]
Isolation of arsenic-resistant bacteria for bioremediation
Microorganisms play a major role in the biochemical cycle
of arsenic and can convert to different oxidation states with
different solubility, mobility and toxicity.[9]
For survival under metal-stressed conditions, bacteria
have evolved several mechanisms to tolerate the uptake of
heavy metal ions. It mainly involves efflux of metal ions
outside the cell, accumulation and complexation of the
metal ions inside the cell and oxidation-reduction of the
heavy metal ions to a less toxic state.[1] Arsenic-resistant
strains also utilizes arsenic in their metabolism, either as a
means of generating energy through chemoautotrophic ar-
senite oxidation[10] or using arsenate as a terminal electron
acceptor in an aerobic respiration.[11,12] These microbes are
widely distributed in the environment and heavy metal con-
taminated soil and water are the potential sources of heavy
metal-resistant bacteria.[8]
Anderson et al. isolated 17 morphologically distinct
arsenic-resistant bacteria that belongs to genera Ex-
iguobacterium, Aeromonas, Bacillus, Pseudomonas, Es-
cherichia, Acinetobacter and all the bacteria showed high
tolerance capacity for arsenic (0–100 mM arsenate or 0–20
mM arsenite).[13] Jackson et al. isolated bacteria that be-
longed to six bacterial groups including the Proteobacte-
ria, Bacteroidetes, and Firmicutes. Some of these bacteria
were capable of growing at high levels of arsenate (up to
275 mM), although arsenite tolerance was much lower (10
mM).[14] Rajdeep et al. isolated Planococcus KRPC10YT
from arsenic-infested bore-well of West Bengal, India and
the bacterium was resistant to exceeding concentrations of
arsenate (30 mM) and arsenite (20 mM).[15] Shivaji et al.
obtained a novel arsenic-resistant bacterium Bacillus ar-
senicus from arsenic sediments in Chakdah district of West
Bengal, India (23◦ 3 N 88◦ 35 E). The bacterium could
grow in the presence of 20 mM arsenate and 0.5 mM ar-
senite.[16]
In the present study, we have described detailed mor-
phological, biochemical characterization and 16S rRNA
analysis of arsenic resistant bacteria isolated from arsenic
contaminated soil of West Bengal, India. The capabil-
ity of the isolates to withstand high concentration of ar-
senic, intracellular accumulation of arsenic and resistance
towards other metal ions were determined. The arsenic-
transforming abilities of the isolates were also studied in
depth with a view to suggesting interventions that will re-
duce pollution in the environment and further the enzymes
produced by these bacteria could be used for development
of commercial products such as biosensors for arsenic.
Materials and methods
Chemicals and stock solutions
Chemicals used in this study were AR grade (Sigma) and
used without further purification. All solutions were pre-
pared with MilliQ (18.2 M) water and were sterilized by
1737
filtration (0.44 µm pore-size) or by autoclave at 120◦ C for
15 min. As [III] stock solutions were prepared freshly before
use from sodium arsenite (NaAsO2 ) and As [V] stock solu-
tions from sodium arsenate (Na2 HAsO4 .7H2 O) and were
stored at 4◦ C in the dark.
Site description and sample collection
Soil samples were collected from arsenic contaminated area
in Chakdah, North 24 Parganas, West Bengal, India (23◦ 39
N, 88◦ 35 E). Subsurface soil (from 0–5 cm in depth) were
collected in sterilized and sealed polythene bags and kept at
4◦ C until further analysis. The pH of soil sample was deter-
mined by shaking 10 g of soil in 20 mL of distilled water for
25 minutes followed by measurement with pH meter (model
Cyberscan PC 510, Eutech Instruments; Netherlands).
Arsenic analysis of the soil sample
The soil samples were air dried and 2.5 g soil sample
was mixed with 1 mL of HNO3 (65 %, Merck) and 3 mL
HCl (37 %, Merck). The mixture was heated to 70◦ C for
1 hour, and then diluted with 5 mL of deionized water.
The acid-digested solution was filtered to remove residual
particulates. Arsenic concentration was determined by
spectrophotometric method[17] using Helios UV-Vis spec-
trophotometer. The results were substantiated by atomic
absorption spectrophotometry and hanging mercury drop
electrode studies.[18]
Isolation of arsenic resistant bacterial strain
One gram of soil sample was dissolved in 10 mL of 0.85 %
sodium chloride solution and shaken for 24 h before iso-
lation in selective media. Samples were diluted 10–10,000
fold with sterile 0.85 % sodium chloride solution and plated
on nutrient agar (NA) plates (0.5 % peptone, 0.5 % NaCl,
0.2 % yeast extract, 0.2 % beef extract and 1.5 % agar, pH
7.0 all w/v) supplemented with 1mM sodium arsenate or
sodium arsenite.
These plates were incubated for 48 hours at 37◦ C. A num-
ber of morphologically different colonies were randomly
picked and isolated after successful purification process in
the same medium. In this preliminary screening, colonies
showing resistance to arsenate or arsenite was selected for
further screening processes.
Culture slants were made and kept at 4◦ C. Dense cell
suspensions were prepared in 10 % (v/v) aqueous glycerol
in one vial and stored at 4◦ C. The pure isolates obtained
were characterized in terms of their morphological, physi-
ological, biochemical and molecular nature.
Morphological characterization
Isolated colonies of purified strains grown on solidified
agar plates were observed and colonial morphology was
1738
recorded.[19] The cellular morphology and Gram’s nature
of the isolates were determined by bright field microscopy
and Scanning Electron Microscopy.[20]
Biochemical characterization
Biochemical tests were performed to determine salt toler-
ance, carbohydrate fermentation, amino acid utilization,
H2 S production, citrate utilization, nitrate reduction abil-
ity and presence of enzymes like oxidase, catalase, lipase,
gelatinase, urease, amylase, etc. Biochemical properties of
the isolates were tested according to Bergey’s Manual of
Systematic Bacteriology.[21]
Physiological characterization
Physiological characterization includes determination of
optimum pH and temperature for growth. The following
areas are included:
Growth at optimum pH and temperature. For all the isolated
bacterial strains, 3 mL of the Luria-Bertani (LB) broth (1
% tryptone, 0.5 % yeast extract and 0.5 % NaCl, pH 7.0 all
w/w) were adjusted at different pH values varied from 2 to
10 and autoclaved at 120◦ C and 105 k Pa for 15 minutes
without metal ion. After 24 h, the growth of the bacteria was
tested through optical density measurement at 600 nm by
UV-Vis spectrophotometer (model Helios # UVG-094737
UV-Visible spectrophotometer, ThermoSpectronic, UK).
Growth at optimum temperature. Tubes of 3 mL LB broth
were equally inoculated with fresh culture and incubated
at 4, 20, 30, 37 and 60◦ C. After 24 h, the growth of the
bacteria was tested through optical density measurement
at 600 nm by UV-Vis spectrophotometer.
PCR amplification and sequencing
Genomic DNA of the isolates was prepared by the sodium
dodecyl sulfate-proteinase K-cetyltrimethylammonium
bromide (CTAB) method. PCR amplification of the
16S rRNA gene fragment was done by using 27F
(5 -AGAGTTTGATCCTGGCTCAG-3 ) and 1492R (5 -
TACGGTTACCTTGTTACGACTT-3 ) primers. The re-
action mixtures composed of 5 µl 10xPCR buffer, 0.2 µM
dNTPs, 1 µM each primer, 5 µl DNA template, 1 units Taq
DNA polymerase and sterile deionized water to a final vol-
ume of 50 µl. PCR profile was as follows: initial denatura-
tion at 96◦ C for 2 min, followed by 35 cycles of denaturation
(96◦ C, 20 sec), annealing (58◦ C, 20 sec), extension (60◦ C, 4
min), and final extension at 72◦ C for 10 min. All the am-
plified PCR products were agarose-gel-eluted by using Hi
Pur A Agarose gel DNA Purification Spin Kit (Catalogue
no. MB511).
The purified gel-eluted rDNA fragments were sequenced
in ABI 3100 Genetic Analyzer with primers: 27F and
1492R. The nucleotide sequences were compared to
Banerjee et al.
published sequences in the NCBI GenBank using the nu-
cleotide BLAST. The sequence being novel was submitted
to GenBank. For phylogenetical analysis, the bacterial
16S rRNA gene sequences from this work and other
sequences retrieved from the database were aligned using
the software CLUSTAL W 1.8.[22] The phylogenetical
analysis was made using the neighbour-joining method.
The analysis was performed with the software BioNJ. The
output trees were prepared using the software Treeview
1.[23] The Biolog system was also used to support the 16s
RNA genus identification of all the isolates.[13]
Evaluation of arsenic resistance
For screening of the isolated strains on the basis of ar-
senic resistivity, 1 % of the overnight grown culture of the
isolates were inoculated in 5 mL of LB broth containing
sodium arsenate and sodium arsenite as sources of the ar-
senic ions (As+5 and As+3). The level of the arsenic tol-
erance of isolated strains was tested by growing them in
different concentrations of sodium arsenate (1–250 mM)
and sodium arsenite (1–100 mM). The growth of the bacte-
ria was tested through optical density measurement at 600
nm and growth was quantified with respect to the control
containing no metals.[24] To evaluate the levels of resistance,
minimum inhibition concentration (MIC) of all the isolates
were determined. The MIC was defined as the lowest con-
centration of arsenite and arsenate that suppressed visible
growth of bacteria.[25]
Effect of arsenic on bacterial growth
For evaluation of the dependence of culture growth on
the presence and absence of metal ions in the media,
the growths of the isolates were determined in the ab-
sence and presence of arsenate or arsenite ions. From
an overnight grown pure culture, 1% innoculum was
added to 50 mL LB broth supplemented with both 5
mM sodium arsenate and 1 mM sodium arsenite and
maintained at optimum pH and was grown at opti-
mum temperature by continuous shaking at 100 rpm in
an orbital shaker. The growth of all the isolates was
monitored by measuring optical density after regular in-
tervals at 600 nm. The doubling time and growth rate con-
stant (k) for the log phase of growth curve was determined
by plotting log10 of the optical density against time.[26]
Samples were collected every 24 hours for the measure-
ment of pH and arsenic concentration of the media. Deter-
mination of arsenic concentration in culture medium was
performed using a spectrophotometric method.[27]
Production of siderophores
The ability of isolates to produce siderophores was checked
by using ferric-perchlorate method.[28] Siderophores are the
Isolation of arsenic-resistant bacteria for bioremediation
low molecular weight, high-affinity chelating agents, which
solubilize ferric iron in the environment and transport it
into the cell.[29] All the isolates were grown on siderophore
production medium (2.5 % Sucrose, 0.4 % (NH4 )2 SO4 , 0.3
% K2 HPO4 , 0.1 % citric acid, 0.008 % MgSO4 , 0.002 %
ZnSO4 , pH 6.8, all w/v) and incubated for 5 days at 30◦ C
and after addition of ferric perchlorate reagent, the forma-
tions of reddish-halos were noted.[28]
Screening of the arsenate-reducing and arsenite-oxidizing
bacteria
The ability of bacterial isolates to oxidize As(III) or reduce
As(V) was tested by the usage of silver nitrate (AgNO3 ).
YEM agar (1 % Mannitol, 0.05 % K2 HPO4 , 0.02 % MgSO4 ,
0.01 % CaCl2 , 0.05 % yeast extract and 1.5 % agar, pH 7.0,
all w/v) containing 1 mM sodium arsenite was used for the
determination of As(III) oxidation and YEM agar contain-
ing 5 mM sodium arsenate was used for the determination
of As(V) reduction. After 5 days of cultivation at 30◦ C, the
agar plates were flooded with 0.1 M AgNO3 solution. The
reaction between AgNO3 and As(III) or As(V) resulted in
the formation of a colored precipitate.[30] A brownish pre-
cipitate revealed the presence of silver arsenate (Ag3 AsO4 )
in the medium (colonies expressing arsenite oxidase), while
a yellow precipitate confirmed the presence of silver arsen-
ite (Ag3 AsO3 ) (colonies expressing arsenate reductase).
Evaluation of metal tolerance
The resistance of the bacterial isolate to various heavy met-
als like NiCl2 , CoCl2 , ZnSO4 , HgCl2 , Pb(NO3 )2 , SbCl3 ,
(NH4 )6 MO7 O24 , CuCl2 , CrCl3 , K2 Cr2 O7 , CdCl2 , SnCl2 ,
AgNO3 , at different concentrations was checked in LB
broth.
Results and discussions
Sample collection and analysis
Soil and effluent water containing elevated concentration of
heavy metals are potential sources of toxic-metal-tolerant
bacteria. It is likely that such environment promotes adap-
tation and selection for heavy metal resistance.[8] For isola-
tion of arsenic resistant bacterial strains, we have collected
soil samples from arsenic contaminated area of North 24
Parganas, West Bengal, India (23◦ 39 N, 88◦ 35 E). The pH
of the soil sample was found to be 7.3, whereas total arsenic
content of the soil was ∼4 mg arsenic/Kg of soil.
Isolation and characterization of arsenic-resistant bacterial
strain
Ten different bacterial strains were isolated from soil sam-
ples and were designated RJB-1, RJB-2, RJB-3, RJB-4,
RJB-5, RJB-6, RJB-A, RJB-B, RJB-C and RJB-D. Out
1739
of these 10 strains RJB-1, RJB-2, RJB-3, RJB-4, RJB-
5 and RJB-6 were isolated on NA medium containing
1 mM sodium arsenate and RJB-A, RJB-B, RJB-C and
RJB-D were isolated on NA medium containing 1 mM
sodium arsenite. The preliminary characterization of the
10 isolates was done on the basis of colonial morphol-
ogy, cellular morphology and gram staining character-
istics.
The colonial morphology of all the strains was studied
in details. Colony shape, elevation, and margin were circu-
lar, convex, and entire type, respectively, for all the strains
except RJB-1, RJB-B and RJB-D which had irregular colo-
nial margins. All 10 bacterial strains were microscopically
studied and cellular morphology such as shape and gram re-
actions were observed during gram staining of each strain.
Cellular shapes of all the strains were found to be bacilli, ex-
cept RJB-6 which was a cocci and all of the isolated strains
were gram negative except RJB-4, RJB-5 and RJB-6 which
were gram positive.
Biochemical characterization was performed in terms of
carbohydrate utilization (starch, dextrose, sucrose, maltose,
rhamnose, fructose, lactose, citrate, etc.), amino acid uti-
lization (glutamic acid, aspartic acid, lysine, phenylalanine,
cysteine), salt tolerance and production of enzymes such as
catalase, oxidase, amylase, urease, DNase, lipase, etc. Table
1 shows the results of various biochemical tests of all the 10
microbial isolates. Biochemical characterization indicated
that RJB-1, RJB-3, RJB-B and RJB-D belong to the genus
Pseudomonas. The detailed biochemical characterization
of the 10 isolates would help to identify the biotechnologi-
cally important strains as well as the enzymes produced by
them can be used for commercial exploitation.
pH plays a major role for the growth and metal accumu-
lation properties of the bacterial strains.[31] The optimum
pH for growth was found to be 7.0 for RJB-1, RJB-3, RJB-
4, RJB-5, RJB-6, RJB-B, RJB-C, RJB-D and 6.0 for RJB-2
and RJB-A. All 10 isolates could tolerate a wide range of
pH from 4–9. Temperature is another environmental fac-
tor that affects bacterial growth.[32] All the isolates showed
growth at 20, 30, 37 and 45◦ C but maximum growth was
observed at 37◦ C.
The 16S rRNA sequences of all 10 isolates were subjected
to nucleotide BLAST and the bacteria were classified ac-
cording to their similarity to sequences in the GenBank
database. All 10 isolates were found to be novel and the
gene sequences were submitted to Genbank (NCBI) and
accession numbers were assigned to all the isolates. Table 2
shows the percentage similarity and accession numbers of
the microbial isolates obtained by blast search at NCBI.
Phylogenetic analysis revealed that RJB-1, RJB-2, RJB-
3, RJB-A, RJB-B, RJB-C and RJB-D belonged to γ -
proteobacteria. RJB-4 and RJB-5 belonged to Firmicutes,
whereas RJB-6 was designated as Kocuria genera. The
phylogenetic tree of 16S rRNA gene sequences showing
the phylogenetic relation between strains RJB-1, RJB-3,
RJB-C and reference species is shown in Figure 1(a) and
1740 Banerjee et al.
Table 1. Biochemical characterization of the 10 bacterial isolates.
Biochemical tests RJB - 1 RJB - 2 RJB – 3 RJB - 4 RJB - 5 RJB – 6 RJB - A RJB-B RJB - C RJB-D

Methyl red − − − + + + − − − −
Nitrate reduction − + − − − + − + +G +
Oxidase + + + − − + − + + +
Catalase + + + − − + + + + +
Voges prausker − − − − − − − − − −
Mannitol salt Agar − − − + + − − − − −
Gelatin hydrolysis − − − − − − − − − −
Starch hydrolysis − − − + + − − − − −
Urea hydrolysis + − + − − + − + + +
H2 S production − − − − − − − − − −
Citrate utilization + + + + + + + + + +
ONPG − − − + + − − − − −
0.8 % NaCl + + + + + + + + + +
Dextrose + − + + + + + + + +
Sucrose − + − + + + + − + −
Maltose − + − + + + + − + −
Rhamnose − − − + + − − − − −
Fructose − − − + + + + − − −
Lactose − + + + + + + + + +
Arabinose + + + + + + + + + +
Sorbitol + + + + + + + + + +
Dulcitol + + + + + + + + + +
Glutamine + + + − − + + + + +
Aspartic acid − − + − − − − − − −
Lysine + − + − − − − − − −
Phenyl alanine − + + − − + + + − −
Cysteine − + − − − − + − − −
∗
“+” denotes positive response, “−” for negative, “+G” positive with gas production.

phylogenetic relation between strains RJB-2, RJB-A and
reference species is shown in Figure 1(b) (all figures not
shown).
Evaluation of arsenic resistance
The resistance to arsenic (arsenate and arsenite) was tested
to determine the potential of the isolated bacteria for biore-
mediation of arsenic. The minimum inhibitory concentra-
tions (MICs) i.e., the lowest concentrations that completely
inhibit bacterial growth[33] were determined experimentally
for all the isolates. Microbial resistance to arsenate or arsen-
ite was determined by visible growth after 24 hours in LB
broth supplemented with varying concentrations of sodium
arsenate or sodium arsenite.
Among 10 arsenic-resistant isolates, RJB-2, RJB-3, RJB-
6, RJB-A, RJB-B and RJB-C exhibited MIC from 100–125
mM of As[V], whereas RJB-1, RJB-4, RJB-5 and RJB-
D exhibited MIC in the range 50–75 mM As[V]. When
the LB broth was supplemented with As[III]), RJB-1,

Table 2. 16S rRNA analysis of the 10 isolates.

Isolate Closest neighbour Percentage similarity Accession number

RJB-1 Pseudomonas aeruginosa 99.56 % FJ756943

RJB-2 Acinetobacter lwoffii 98.53 % FJ756944
RJB-3 Pseudomonas resinovorans 98.53 % FJ866631
RJB-4 Bacillus circulans 97.76 % FJ756945
RJB-5 Bacillus circulans 98.51 % FJ756946
RJB-6 Kocuria palustris 99.78 % FJ756947
RJB-A Acinetobacter calcoaceticus 99.12 % FJ866632
RJB-B Pseudomonas alcaligenes 99.7 % FJ866633
RJB-C Vogesella indigofera 99.76 % FJ756948
RJB-D Pseudomonas alcaligenes 99.0 % FJ756949
Isolation of arsenic-resistant bacteria for bioremediation 1741

Fig. 1. Phylogenetic tree based on 16S rRNA gene sequences, showing the relationship between strain (a)RJB-1, RJB-3, RJB-C and
related bacteria. (b) RJB-2, RJB-A and related bacteria. The branching pattern was generated by the neighbour-joining method.
Based on 1,000 replications, bootstrap percentages above 50 % are shown (color figure available online).

RJB-3 and RJB-C showed MIC in the range 10–25 mM, but Effect of arsenic on bacterial growth
RJB-2 and RJB-A demonstrated a higher MIC of 50 mM.
To determine the effects of As[V] and As[III] ions on bacte-
Results of MIC are shown in Table 4. Most of the bacterial
rial growth, all the bacterial isolates were grown in arsenic-
isolates showed high levels of MIC, which may be due to the
free media and media supplemented with 5 mM and 1
environmental factors or genetic structure of the bacteria.
mM arsenic as arsenate and arsenite ions, respectively. The
The higher values of MIC for arsenic (as As[V] and As[III])
growth of the isolates was determined by measurement of
indicated that some of these bacteria could be utilized for
optical density at regular intervals. While observing the
bioremediation purposes.
growth, pH change of the media and arsenic uptake by the
1742 Banerjee et al.
Table 3. Doubling time and growth rate constant of the isolates in presence and absence of arsenic.

Doubling time (hour) Growth rate constant (h−1)

Isolate LB As[V] As[III] LB As[V] As[III]

RJB-1 3.524 3.924 8.819 0.197 0.177 0.079

RJB-2 10.948 10.231 10.090 0.063 0.068 0.069
RJB-3 5.738 5.707 9.720 0.121 0.121 0.071
RJB-4 1.248 1.241 5.020 0.555 0.558 0.138
RJB-5 1.265 1.375 5.695 0.547 0.503 0.122
RJB-6 4.66 4.856 5.772 0.148 0.142 0.12
RJB-A 11.191 12.597 13.105 0.062 0.055 0.053
RJB-B 2.431 3.163 4.732 0.285 0.219 0.146
RJB-C 2.629 2.280 7.886 0.264 0.304 0.088
RJB-D 3.353 2.668 5.78 0.206 0.26 0.119

bacterial cells were also monitored. All the isolates follow
a sigmoid pattern of growth.
It was observed that strain RJB-1 grew very well in the
presence of As[V] in comparison to its growth in presence
of As[III]. The growth rate constant (k) calculated in the
absence of arsenic was 0.2 h−1 (a doubling time of 3.52 h);
in the presence of As[V] it was 0.18 h−1 (a doubling time
of 3.92 h), and in the presence of As[III] it was 0.08 h−1 (a
doubling time of 8.82 h). The data suggested 60 % reduction
in the cellular growth of the isolate in presence of As[III]
ions. It was also found that As[V] reduction by microbes
was coupled to alkalinization of the growth medium. The
pH of the As[V] medium was found to increase gradually
from 7.0 to 9.2 from lag to stationary phase, while the pH
change increased from 7.0 to 8.8 in As[III] media. After
10 days of incubation, RJB-1 showed decrease in As[V]
content from 5 mM to 2.1 mM and As[III] content from 1
mM to 0.66 mM (Fig. 2).
Isolate RJB-2 did not show any significant differences
in there growth curve in the presence and absence of ar-
senic. The growth rate constant calculated in the absence
of arsenic was 0.063h−1 (a doubling time of 10.95 h); in
the presence of As[V] it was 0.068 h−1 (a doubling time
of 10.23 h), and in the presence of As[III] it was 0.07 h−1
(a doubling time of 10.09 h). The pH of As[V] medium
changed from 7.0 to 9.0, while the pH change increased
from 7.0 to 8.5 for As[III]-medium. RJB-2 demonstrated
decrease in As[V] content of 3.19 mM and As[III] content
of 0.972 mM after 10 days of incubation (Fig. 3).
Isolate RJB-A exhibited same pattern of growth in pres-
ence and absence of arsenic. The growth rate constant cal-
culated in the absence of arsenic was 0.062 h−1 (a doubling
time of 11.19 h), in the presence of As[V] it was 0.055 h−1
(a doubling time of 12.6 h), and in the presence of As[III]
it was 0.053h−1 (a doubling time of 13.11 h). The pH of the
medium was found to increase gradually to 8.35 and 7.7 for
As[V]- and As[III]-containing medium, respectively. After
10 days of incubation, RJB-A reduced As[V] concentration
to 3.3 mM and As[III] concentration to 0.94 mM (Fig. 4).
The isolates RJB-3 and RJB-C exhibited very poor
growth in presence of As[III] compared to the growth
curves of arsenic-free and As[V]-containing media. A re-
duction of 41.6 % and 65.3 % was observed in the cellu-
lar growth of the bacterial isolate in presence of arsenite
ions. The pH change of the As[V] media increased to 8.33
whereas the pH of the As[III] media increased slightly to

Table 4. MIC, arsenic transforming ability and siderophore production of the arsenic-resistant isolates.
Minimum inhibitory
concentration (mM)
Bacterial isolates Arsenate Arsenite Arsenate reducing Arsenite oxidizing Siderophores

RJB-1 50 10 + − +
RJB-2 125 50 + + +
RJB-3 120 20 + − +
RJB-4 75 20 + + −
RJB-5 75 50 + + −
RJB-6 100 25 − − −
RJB-A 125 25 + + +
RJB-B 125 25 − − −
RJB-C 100 25 + − −
RJB-D 75 15 − − −
Isolation of arsenic-resistant bacteria for bioremediation 1743

Fig. 2. (a) Growth pattern and extracellular pH change of isolate RJB-1 in absence and presence of arsenate ions (5 mM) and arsenite
ions (1 mM), and (b) arsenic removal and uptake capacity of the isolates RJB-1 in presence of arsenate ions (5 mM) and arsenite ions
(1 mM).

Fig. 3. (a) Growth pattern and extracellular pH change of isolate RJB-2 in absence and presence of arsenate ions (5 mM) and arsenite
ions (1mM), and (b) arsenic removal and uptake capacity of the isolates RJB-2 in presence of arsenate ions (5 mM) and arsenite ions
(1 mM).

Fig. 4. (a) Growth pattern and extracellular pH change of isolate RJB-A in absence and presence of arsenate ions (5 mM) and arsenite
ions (1 mM), and (b) arsenic removal and uptake capacity of the isolates RJB-A in presence of arsenate ions (5 mM) and arsenite
ions (1 mM).
1744 Banerjee et al.

Fig. 5. Scanning electron micrograph depicting effect of metals on cellular morphology of the isolates (a) Isolate RJB-2 in absence
of arsenic, (b) Isolate RJB-2 in presence of arsenic, Transmission electron micrograph of (c) RJB-2 in presence of arsenic, and (d)
EDAX analysis of RJB-2 in presence of arsenic (color figure available online).

7.55 during growth of RJB-3. In the case of RJB-C, the
pH of As[V] media increased to 8.6 and of As[III] media to
7.14. RJB-3 also displayed removal of 3.28 mM As[V] and
0.95 mM As[III], RJB-C displayed removal of 3.48 mM
and 0.91 mM for As[V] and As[III], respectively.
The growth pattern of isolates RJB-4, RJB-5, RJB-6,
RJB-B and RJB-D showed that they could grow very well in
absence of arsenic and presence of As[V] in comparison to
growth pattern in presence of As[III] ions. The growth rate
constant and doubling time of all the 10 arsenic-resistant
bacterial isolates are shown in Table 3. The alkalization
of the As[V] medium, in case of all the bacteria, might
be due to the reduction of As[V] to As[III], whereas the
bacteria growing in As[III] medium was not found to alter
the pH of the medium. This overall study implies that all the
bacterial isolates could very well grow in presence of As[V]
whereas some of the strains (RJB-4, RJB-5, RJB-6, RJB-B
and RJB-D) could grow well in presence of As[III]. The
isolates RJB-1, RJB-2, RJB-3, RJB-A and RJB-C showed
greater arsenic (both As[III] and As[V]) removal efficiency
compared to the other bacterial isolates and could therefore
be used for bioremediation of arsenic.
SEM, TEM and EDAX analysis
Microscopic studies and SEM images of the bacterial iso-
lates grown in the presence and absence of arsenic indi-
cated that RJB-2, RJB-3 and RJB-A formed long chains in
Isolation of arsenic-resistant bacteria for bioremediation 1745
Table 5. MIC values for different metals and metalloids.
MIC of bacterial isolates (mM)
Metals RJB-1 RJB-2 RJB-3 RJB-4 RJB-5 RJB-6 RJB-A RJB-B RJB-C RJB-D

Nickel 25 10 25 20 10 10 10 20 20 20
Cobalt 10 10 15 15 10 5 3 10 5 10
Zinc 15 25 20 30 30 30 25 25 15 25
Mercury − − − − − − − − − −
Lead 25 30 25 30 30 25 20 20 15 25
Antimony − 15 7 5 7 5 20 10 − 5
Copper 20 30 25 30 30 25 25 30 15 25
Chromium(III) 25 10 10 10 15 10 20 − 10 10
Chromium(VI) 20 10 15 15 15 20 10 10 25 10
Cadmium 5 3 2 − − − 10 − 5 −
Tin 5 10 7 5 5 5 7 7 7 10
Silver − − − − − − − − − −
“−” denotes “no growth.”

presence of arsenic (both As[III] and As[V]) compared Production of siderophores

to the untreated cells (Figs. 5a and 5b). This chainlike
structure depicts mode of response to metal stress. On the
other hand, isolates RJB-1, RJB-4, RJB-5 and RJB-C re-
duced their sizes when grown in presence of arsenic (both
As[III] and As[V]). These changes in morphological struc-
ture might be a possible stratergy of cells to accumulate
metals. Transmission electron microscopy was also used to
locate intracellular accumulation of arsenic within the bac-
terial cell. TEM results indicated presence of electron dense
deposits throughout the cytoplasm of all the bacterial iso-
lates (Fig. 5c).
The presence of arsenic was also confirmed by EDAX
analysis (Fig. 5d). The study of intracellular accumulation
of arsenic, indicated that most of bacterial strains (except
RJB-B and RJB-D) could accumulate arsenic and con-
tribute in bioremediation of arsenic. The accumulation of
arsenic mainly takes place due to adsorption of the neg-
atively charged arsenic ions by oppositely charged amino
groups in the bacterial cell walls[34], methylation followed
by reduction and oxidation of As ions[35] or sequestration
by a range of cysteine-rich peptides.[36]
Screening of the arsenic-resistant bacteria
A qualitative silver nitrate (AgNO3 ) screening technique
was used to detect the oxidation of As[III] to As[V] or the
reduction of As[V] to As[III]. The interaction of AgNO3
generates bright yellow precipitate and brown precipitate
with As[III] and As[V], respectively. According to AgNO3
test, the strains RJB-2, RJB-4, RJB-5 and RJB-A demon-
strated oxidizing as well as reducing abilities to arsenic. For
all the isolates, the intensity of As[III]-oxidation was less
compared to As[V]-reduction. RJB-1, RJB-3 and RJB-C
showed the ability to reduce As[V] (Table 4). The AgNO3
test revealed that some the isolates could produce both ar-
senite oxidase and arsenate reductase enzymes, which could
be further purified for commercial use e.g., construction of
arsenic biosensor.
Ten isolates were tested for production of siderophores
and it was observed that the bacteria showing highest ar-
senic resistance (RJB-1, RJB-2, RJB-3 and RJB-A) pro-
duced siderophores (shown in Table 4). Microorganisms
produce siderophores that play an important role in iron
(ferric and ferrous ions) uptake from insoluble minerals.
During this process, arsenic ions are mobilized from the
solid (rocks/minerals) to the aqueous phase and the ions
can therefore, easily enter within the cell. This increases the
intracellular cellular uptake of arsenic ions.[37] Thus, the
study of siderophore production showed a new approach
towards the application of the isolated bacterial strains for
bioremediation.
Evaluation of metal resitance
In addition to arsenic resistance, all the isolates could very
well grow in presence of different metals and metalloids.
Multiresistance to Ni, Zn, Pb, Cu, Cr(III) and Cr(VI) was
observed in all the 10 isolates and exhibited MIC in the
range 10–30 mM. They showed minimum tolerance for Co,
Sb, Cd and Sn and could not grow in presence of Hg and Ag.
MICs for different metals are shown in Table 5. It is known
that genetic determinants for heavy metal resistance are
widespread in microbes,[38] and without selective pressure
they are often present in bacterial genomes.[37] Therefore,
these bacterial isolates can be utilized for bioremediation
of soil/effluents containing other metal ions.
Conclusions
In this study, 10 different bacterial strains were iso-
lated and detailed morphological, biochemical, physiolog-
ical and molecular characterizations of these strains were
accomplished. The study specifically indicated application
1746
of the isolates for different purposes. The qualitative screen-
ing of arsenate-reducing and arsenite-oxidizing bacteria
showed a new approach towards bioremediation of arsenic
using bacteria. Arsenite-oxidizing bacteria converts arsen-
ite (toxic) to arsenate (less toxic form), whereas arsenate-
reducing bacteria converts arsenate to arsenite. In both the
cases, a significant portion of arsenate and arsenite con-
verted by bacteria was not extruded out of the cell. The
TEM study also proved that there was intracellular accu-
mulation of arsenic within the bacterial cell.
All these studies suggested that these bacteria did not re-
lease arsenic after altering the oxidation states. Therefore,
this phenomenon would be the most important finding in
favor of a bioremediation by the microbes. High tolerance
(MIC) and arsenic uptake capacity study of the isolates in-
dicated that these bacterial isolates could be suitably used
for development of bioremediation processes. The toler-
ances of the bacteria in presence of many other potentially
hazardous metal ions would make them suitable for biore-
mediation of soil and effluents. The detailed biochemical
characterization shows that all these bacteria can be a po-
tent source of various biotechnologically important and
commercially available enzymes.
Acknowledgment
The authors gratefully acknowledge financial support
from DST, Government of India through Sensor-Hub
at CGCRI, Kolkata, India. The author SB is grate-
ful to CSIR, India for providing fellowship support
(09/028(0772)/2010-EMR-I).
References
[1] Nies, D.H. Microbial heavy metal resistance. Appl. Microbiol.
Biotechnol. 1999, 51, 730–750.
[2] USEPA. The Hazardous Waste System, Environmental Protection
Agency. Washington, DC: Office of Solid Waste and Emergency
Response; 1987.
[3] Cullen, W.R.; Reimer, K.J. Arsenic speciation in the environment.
Chem. Rev. 1989, 89, 713–764.
[4] Ehrlich, H.L. Geomicrobial interactions with arsenic and antimony.
In: Ehrlich H.L. (ed). Geomicrobiology, 3rd ed., New York: Marcel
Dekker Inc.; 1996, pp. 276–293.
[5] Ordon∼ez, E.; Letek, M.; Valbuena, N.; Gil, J.A.; Mateos, L.M.
Analysis of genes involved in arsenic resistance in Corynebacterium
glutamicum ATCC 13032. Appl. Environ. Microbiol. 2005, 71,
6206–6215.
[6] Chakraborti, D.; Das, B.; Rahman, M.M.; Chowdhury, U.K.;
Biswas, B.; Goswami, A.B.; Nayak, B.; Pal, A.; Sengupta, M.K.;
Ahamed, S.; Hossain, A.; Basu, G.; Roychowdhury, T.; Das, D.
Status of groundwater arsenic contamination in the state of West
Bengal, India: A 20-year study report. Mol. Nutr. Food. Res. 2009,
53, 542–551.
[7] Chaalal, O.; Zekri, A.Y.; Islam, R. Uptake of heavy metals by mi-
croorganisms: an experimental approach. Energy Sources 2005, 27,
87–100.
[8] Clausen, C.A. Isolating metal-tolerant bacteria capable of removing
copper, chromium, and arsenic from treated wood. Waste Manage.
Res. 2000, 18, 264–268.
Banerjee et al.
[9] Silver, S.; Phung, T. Genes and enzymes involved in bacterial oxida-
tion and reduction of inorganic arsenic. Appl. Environ. Microbiol.
2005, 71, 599–608.
[10] Santini, J.M.; Sly L.I.; Schnagl R.D.; Macy, J.M. A new
chemolithoautotrophic arsenite-oxidizing bacterium isolated from
a gold mine: Phylogenetic, physiological and preliminary bio-
chemical studies. Appl. Environ. Microbiol. 2000, 66, 92–
97.
[11] Ahmann, D.; Roberts A.L.; Krumholz, L.R.; Morel, F.M.M. Mi-
crobe grows by reducing arsenic. Nature 1994, 27, 371–750.
[12] Stolz, J.F.; Oremland, R.S. Bacterial respiration of arsenic and sele-
nium. FEMS Microbiol. Rev. 1999, 23, 615–627.
[13] Anderson, C.R.; Cook, G.M. Isolation and characterization of
arsenate-reducing bacteria from arsenic contaminated sites in New
Zealand. Curr. Microbiol. 2004, 48, 341–347.
[14] Jackson, C.R.; Dugas, S.L.; Harrison, K.G. Enumeration and char-
acterization of arsenate-resistant bacteria in arsenic free soils. Soil
Biol. Biochem. 2005, 37, 2319–2322.
[15] Chowdhury, R.; Sen, A.K.; Karak, P.; Chatterjee, R.; Giri, A.K.;
Chaudhuri, K. Isolation and characterization of an arsenic-resistant
bacterium from a bore-well in West Bengal, India. Ann. Microbiol.
2009, 59, 253–258.
[16] Shivaji, S.; Suresh, K.; Chaturvedi, P.; Dubeand, S.; Sengupta, S.
Bacillus arsenicus sp. nov., an arsenic-resistant bacterium isolated
from a siderite concretion in West Bengal. India. Int. J. Syst. Evol.
Microbiol. 2005, 55, 1123–1127.
[17] Johnson, D.L.; Pilson, M.E.Q. Spectrophotometric determination
of arsenite, arsenate, and phosphate in natural waters. Anal. Chim.
Acta 1972, 58, 289–299.
[18] Pal, P.; Sarkar, P.; Bhattacharyay, D.; Pal, S. Development of some
electrochemical systems for detection of arsenic in drinking water.
Sensor Lett. 2010, 8, 577–583.
[19] Pelczar, M.J.J.; Reid, R.D. Pure cultures and growth characteristics.
In: Microbiology. McGraw-Hill Book Company, New York; 1958:
pp. 76–84.
[20] Eichorst, S.A.; Breznak, J.A.; Schmidt, T.M. Isolation and char-
acterization of soil bacteria that define Terriglobus gen. nov., in
the Phylum Acidobacteria. Appl. Environ. Microbiol. 2007, 73,
2708–2717.
[21] Krieg, N.R. Bergey’s Manual of Systematic Bacteriology, Vol 1.
Baltimore, MD: Williams and Wilkins; 1984.
[22] Salomon, P.S.; Janson, S.; Granéli, E. Molecular identification of
bacteria associated with filaments of Nodularia spumigena and
their effect on the-cyanobacterial growth. Harmful Algae 2003, 2,
261–272.
[23] Page, R.D.M. TreeView: An application to display phylogenetic
trees on personal computers. Comp. Appl. Biosci. 1996, 12, 357–358.
[24] Hussein, H.; Farag, S.; Moawad, H. Isolation and characterization
of Pseudomonas resistant to heavy metals contaminants. Arab. J.
Biotechnol. 2004, 7, 13–22.
[25] Courvalin, P.; Goldstein, F.; Philippon, A.; Sirot, J.
L’Antibiogramme. MPC-Videom (Paris); 1985.
[26] Pirt, S.J. Principles of Microbe and Cell Cultivation. Oxford: Black-
well: 1975.
[27] Dhar, R.K.; Zheng, Y.; Rubenstone, J.; van Geen, A. A rapid colori-
metric method of measuring arsenic concentrations in groundwater.
Anal. Chim. Acta 2004, 526, 203–209.
[28] Calvente, V.; de Orellano, M.E.; Sansone, G.; Benuzzi, D.;
Sanz de Tosetti, M.I. A simple agar plate assay for screening
siderophore producer yeasts. J. Microbiol. Meth. 2001, 47, 273–
279.
[29] Neilands, J.B. Siderophores. Arch. Biochem. Biophys. 1993, 302,
1–3.
[30] Simeonova, D.; Lievremon,t D.; Lagarde, F.; Muller, D.; Groudeva,
V.; Lett, M.C. Microplate screening assay for detection of arsenite-
oxidizing and arsenate-reducing bacteria. FEMS Microbiol. Lett.
2004, 237, 249–253.
Isolation of arsenic-resistant bacteria for bioremediation
[31] Doenmez, G.; Aksu, Z. Bioaccumulation of copper (II) and Nickel
(II) by the non adapted and adapted growing Candida sp. Water
Res. 2001, 35, 1425–1434.
[32] Bhakoo, M.; Herbert, R.A. The effects of temperature on
the fatty acid and phospholipid composition of four obli-
gately psychrophilic Vibro spp. Arch. Microbiol. 1979, 121, 121–
127.
[33] Muller, D.; Lievremont, D.; Simeonova, D.D.; Hubert, J.C.;
Lett, M.C. Arsenite oxidase aox genes from a metal-
resistant beta-proteobacterium. J. Bacteriol. 2003, 185, 135–
141.
[34] Bai, R.S.; Abraham, T.E. Studies on chromium (VI) adsorption-
desorption using immobilized fungal biomass. Bioresour. Technol.
2003, 87, 17–26.
1747
[35] Brunken, H.S.; Nehrhorn, A.; Breunig, H.J. Isolation and character-
ization of a new arsenic methylating bacterium from soil. Microbiol.
Res. 1996, 151, 37–41.
[36] Dhankher, O.M.; Li, Y.; Rosen, B.P.; Shi, J.; Salt, D.; Senecoff, J.F.;
Sashti, N.A.; Meagher, R.B. Engineering tolerance and hyperac-
cumulation of arsenic in plants by combining arsenate reductase
and γ -glutamylcysteine synthetase expression. Nature. Biotechnol.
2002, 20, 1140–1145.
[37] Drewniak, L.; Styczek, A.; Majder-Lopatka, M.; Sklodowska, A.
Bacteria, hypertolerant to arsenic in the rocks of an ancient gold
mine, and their potential role in dissemination of arsenic pollution.
Environ. Pollut. 2008, 156, 1069–1074.
[38] Silver, S. Bacterial resistances to toxic metals–A review. Gene 1996,
179, 9–19.