Korean J. Microbiol. Biotechnol.

(2014), 42(3), 238248

http://dx.doi.org/10.4014/kjmb.1401.01004
pISSN 1598-642X eISSN 2234-7305

Korean Journal of Microbiology and Biotechnology

Potential for the Uptake and Removal of Arsenic [As (V) and
As (III)] and the Reduction of As (V) to As (III) by Bacillus
licheniformis (DAS1) under Different Stresses
Kumari Tripti1, D. Sayantan1, Shardendu Shardendu1*, Durgesh Narain Singh2, and Anil K. Tripathi2
1
Laboratory of Environment and Biotechnology, Department of Botany, Patna Science College, Patna University,
Patna 800005, India
2
School of Biotechnology, Banaras Hindu University, Varanasi 221005, India

Received: January 27, 2014 / Revised: April 3, 2014 / Accepted: May 22, 2014

The metalloid arsenic (Z = 33) is considered to be a significant potential threat to human health due to its ubiquity and toxicity,
even in rural regions. In this study a rural region contaminated with arsenic, located at longitude 85 32'E and latitude 25 11'N,
was initially examined. Arsenic tolerant bacteria from the rhizosphere of Amaranthas viridis were found and identified as Bacillus licheniformis through 16S rRNA gene sequencing. The potential for the uptake and removal of arsenic at 3, 6 and 9 mM
[As(V)], and 2, 4 and 6 mM [As(III)], and for the reduction of the above concentrations of As(V) to As(III) by the Bacillus licheniformis were then assessed. The minimal inhibitory concentrations (MIC) for As(V) and As(III) was determined to be 10 and 7
mM, respectively. At 3 mM 100% As(V) was uptaken by the bacteria with the liberation of 42% As(III) into the medium, whereas
at 6 mM As(V), 76% AS(V) was removed from the media and 56% was reduced to As(III). At 2 mM As(III), the bacteria consumed 100%, whereas at 6 mM, the As(III) consumption was only 40%. The role of pH was significant for the speciation, availability and toxicity of the arsenic, which was measured as the variation in growth, uptake and content of cell protein. Both As(V)
and As(III) were most toxic at around a neutral pH, whereas both acidic and basic pH favored growth, but at variable levels.
Contrary to many reports, the total cell protein content in the bacteria was enhanced by both As(V) and As(III) stress.
Keywords: Arsenate [As(V)], arsenite [As(III)], Bacillus licheniformis, reduction, toxicity, uptake

Introduction
Arsenic is an ubiquitous metalloid and its compounds are
common in many environmental compartments near toxic
level. Natural sources of arsenic are minerals, found commonly concentrated around sulphide bearing minerals and
hydrous iron oxides [24]. Other sources are reported from
anthropogenic activities like use of pesticides, burning of
coal, industrial metal smelting etc. In environment, inorganic arsenic occurs in many oxidations states those are

*Corresponding author
Tel: +919473240391, Fax: +916122670877
E-mail: shardendu77@rediffmail.com
2014, The Korean Society for Microbiology and Biotechnology

http://dx.doi.org/10.4014/kjmb.1401.01004

As(V) arsenate, As(III) arsenite, As(0) elemental arsenic,

As(-III) arsenide. Among them trivalent and pentavalent
forms of arsenic are most common but As(III) is most toxic.
Arsenic is considered the most significant potential threat
to human health due to its ubiquity and toxicity. Hence
removal of arsenic from environment is of great importance
for human welfare. To trace out, the occurrence and distribution of native flora capable of arsenic tolerance is very
important in bacterial biotechnology. It makes us to understand the extent of potential of such flora in detoxifying
arsenic through oxidation, reduction or methylation [1].
Indo-Gangetic plain of Bengal delta, which includes Bihar
is a principle arsenic sink. Agriculture is the major source of
livelihood for majority of population of this region. Arsenic
contaminated ground water is used for the irrigation, which

Uptake/removal and Reduction of As Species by B. licheniformis

is taken by crops and from this trophic level arsenic enters

into food chain and cause many health hazards. Rapid and
huge withdrawal of water creates an opportunity to release
the arsenic from its chelated sources. Hence it is of immediate importance that natural bacterial flora need to be isolated and its potential for remediation and biotransformation
need to be work out.
Relative abundance of As(V) and As(III) in soil environment is influenced by microbial transformations. Bacteria
developed various strategies to resist arsenic toxicity either
by extrusion after oxidation or reduction of arsenic species
or by intracellular chelations.
For uptake and transport by bacteria, both As(V) and
As(III) are available at neutral pH [44]. As(V) are found in
ionised form as an oxyanion (AsO43) at neutral pH and are
transported into bacterial cell by phosphate (PO43) transporters due to chemical analogy with phosphate. As(V) can
also easily incorporated into metabolic pathway of an
organism as a substitute of phosphate [46]. This substitution for phosphate and inhibition of oxidative phosphorylation is the main toxic effects of As(V) [17]. As(III) are found
mostly unionised as As(OH)3 at neutral pH and are transported into cells by aqua glycoporins (glycerol transport
proteins) [36, 38]. As(III) has high affinity for cells protein
thiol so it can easily chelated with protein and result toxicity.
There are two systems in cell, which help in tolerating
arsenate As(V). The first system is to uptake As(V) with the
help of transporters and reduce into As(III) with the help of
enzyme arsenate reductase. The second system help to
extrude reduced As(III) from cell. Arsenic reducing bacteria
contain arsenic resistance gene (ars) either on chromosome or on plasmid, which confers arsenic resistance by
encoding two necessary components (a) reductase enzyme
(ArsC), for reducing arsenic(V) to arsenic(III). (b) an efflux
pump (ArsB) that extrudes As(III) from the cytoplasm for
lowering intracellular concentration of toxic arsenic [37].
As(III) uptake occurs by glycerol transporter and get chelated with intracellular protein by thiols.
Protein content of bacteria under different stress are variable [41]. The goal of present study is to isolate and identify
the arsenic resistant bacteria and evaluation of the potential
to tolerate As(V) and As(III). The other objective is to determine the potential of reduction of As(V) to As(III). The
effects of pH on growth and uptake have also been determined, total protein content of bacterial cell has estimated
to check the level of stress.

239

Material and Methods

Isolation of bacteria
In natural habitat, bacteria grow together containing
many species and to study on the individual bacteria one
need a pure culture of a single strain. Spread plate technique [35] had been used to obtain a pure culture of a single strain of bacteria. Soil was isolated from rhizosphere of
Amaranthas viridis, which was grown on arsenic contaminated area. 1 mg of soil sample was taken and diluted with
sterile water. 1 ml of diluted soil was put on tryptone yeast
extract glucose (TYEG) [33]. As living cell, generally
exposed to arsenic in the form of arsenate and arsenite in
which As(V) are most available, hence arsenate has been
used for isolation of arsenic resistant bacteria. Agar plates
amended with 10 M, 100 M, 1 mM and 10 mM of arsenate (Sodium arsenate Na2HAsO47H2O) and spread
evenly over the surface with the help of sterile L-shaped
glass rod. Plates were incubated at 37C for 24 h. Many
visible bacterial colonies were grown on plates; only 10 mM
plates contain no colony. Hence one of the isolate, which
was able to grow on 1 mM As (V) amended TYEG plate
was considered as more tolerant species and was selected
for further study.
Growth phase study and minimum inhibitory concentration (MIC)
Overnight grown culture was prepared in 50 ml of TYEG
broth. For subculturing, into test and control tubes 0.5 ml of
bacterial culture was aseptically transferred into culture
tubes containing 4.5 ml of TYEG broth, which had been
previously amended with arsenic (Control no added arsenic).
The culture tubes were then incubated at 37C and
150 rpm in shaker incubator. At different time interval, from
0, 1, 2, 4, 6, 8, 10, 24, 26, 28, 30 h, cultures were taken for
turbidity measurement. Absorbance was measured at
600 nm in (Double beam spectrophotometer, Systronics,
India); uninnoculated broth was served as a blank. In one
set of experiment, broth amended by one particular concentration of As(V) or As(III) and the same procedure was
repeated for various concentration until the MIC obtained.
Estimation of concentration of As(V) and As(III) in residual media and As in biomass
Bacterial cultures prepared for turbidity measurement at
various time intervals were also taken for estimation of

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Tripti et al.

amount of arsenic in residual media. Bacterial culture were

taken in clean sterile Eppendorf tubes (1.5 ml) and centrifuged at 10,000 rpm for 2-4 min. Pellet were taken separately for biomass digestion [16] and 1 ml of cell free
supernatant was taken into clean culture tubes with 9 ml of
distilled water and subjected to digest by nitric acid method
[2]. Before digestion, diluted supernatant was taken for estimation of amount of As(III) in residual media by Azure B
method [9]. After digestion, sample was further allowed to
estimate for total arsenic in residual media by Azure B
method with some modifications. Concentration of As(V)
obtained by subtracting concentration of As(III) from concentration of total arsenic in medium. Pellet obtained above
were washed twice and placed in an oven for drying and
dried sample was digested by nitric acid method and then
arsenic present in biomass was estimated by Azure B
method.
Extraction and estimation of bacterial cell protein
5 ml bacterial broth culture was prepared in culture tubes
amended with arsenate As(V),and As(III) with acidic and
basic pH. It was observed that pH of normal TYEG broth
was 7.2 i.e. approximately 7, pH of arsenate/As(V) and
arsenite/As(III) enriched media was 7.8 and 8.7, respectively. Hence pH 6 as (acidic pH) and pH 9 and 10 as (basic
pH) taken for arsenate and arsenite stress, respectively.
Bacterial cultures at different time intervals were taken for
protein extraction. Bacterial culture was taken in clean sterile Eppendorf tubes (1.5 ml) and centrifuged at 12,000 rpm
for 10 min at 10C. Cell free supernatant was discarded
and to the pellet 50 l of lysozyme (10 mg/ml) was added
and kept for incubation at 37C for 20 min [45] after that
cells were vortexed and again centrifuged at 12,000 rpm for
10 min. Supernatant was transferred into clean test tubes
and volume was make up with distilled water upto 1 ml. 5
ml of Coomassie reagent was added into each test tube
and after incubation reading was taken at 595 nm [7] in
Double beam Spectrophotometer. Amount of protein calculated with the help of protein SDR prepared according to
Bradford protocol [7].
Identification of isolates based on 16S rRNA gene
sequence
PCR amplification: Total genomic DNA of the isolate
was extracted with Wizard genomic DNA purification kit

http://dx.doi.org/10.4014/kjmb.1401.01004

(Promega, Madison, WI, USA). 16S rRNA gene was amplified using universal bacterial primer 8f (5'-AGA GTT TGA
TYM TGG CTC AG- 3' and 1495r (5'-CTA CGG CTA CCT
TGT TAC G-3') [18]. A 50 l reaction mixture was prepared,
which include 50 ng of bacterial DNA as template, 200250 M of each primer, and 1.0 unit of Taq DNA polymerase (Genei, Bangalore). The Polymerase chain reactions were performed on Veriti 96 well Thermal cycler
(Applied Biosystem, USA) under the reaction condition of
an initial denaturation of 5 min at 95oC followed by 35
cycles of 1 min at 94oC, 1 min at 51oC, and 1 min at 72oC,
with a final extension of 5 min at 72oC. The 16S rRNA gene
amplicons were analyzed in 0.8% agarose gel at 5 V cm1
and visualized under UV light with Alpha imager (Alpha
Innotech Corporation, UK).
Sequencing: 16S rRNA genes were purified with Wizard
SV gel PCR purification kit (Promega, USA) and quantified
using ND-1000 Spectrophotometer (NanoDrop, Wilmington, USA). Direct sequencing was performed at SBT, BHU
with three primers 8f (5'-AGA GTT TGA TYM TGG CTC
AG-3'), 1495r (5'-CTA CGG CTA CCT TGT TAC G -3') and
561f (5'-AATTACTGGGCGTAAAG-3') [8] using the BigDye
Terminator v3.1 Cycle sequencing kit (Applied Biosystems,
Switzerland) in an ABI PrismTM 310 automated DNA
Sequencer (Applied Biosystems).
Analysis of 16S rRNA gene sequence: 16S rRNA
gene sequences were edited using Bioedit software version
3.1 to make a complete sequence. The almost complete
sequence was compared with the nucleotide sequences
present in the NCBI database using the standard nucleotide BLAST search.
Gene Bank accession numbers: The nucleotide
sequence of 16S rRNA gene of isolate has been deposited
in Gene Bank under the accession number KF664027.
Data analysis: All the data in the figures are the
means standard errors of three replicates. The correlation
between As(V) uptake and As(V) reduction to As(III) were
expressed as scattered plot. The significance of effect of
pH on uptake/removal of As(V) and As(III) was calculated
by Students t-test. The significance of variation of cell protein content of bacterial cell in different stress was also calculated by Students t-test. The statistical evaluation were
performed by the software STATISTICA5.52.164.0 and the
graphs were drawn using MICROSOFT EXCEL 2003,
2007.

Uptake/removal and Reduction of As Species by B. licheniformis

241

Results and Discussion

other member of bacillus species.

Isolation of arsenic resistant bacteria

Arsenic contamination is intensified by change in geochemical cycles resulted from anthropogenic activities [43]
and also increased by microbial metabolism [15, 22].
Above phenomena resulted the release of arsenic into
drinking water in shallow wells. Ground water of Indo-Gangetic plain of Bengal Delta (Bihar located at 85 32'E longitude and 25 11'N latitude on the Earth) has been reported
as contaminated by arsenic [10, 11]. Identification of native
bacterial flora, with the assessment of their potential of
uptake/removal of arsenic species and its biotransformation, will be an addition of novel result in research data as
the first this type of report from above biotope of the earth.
Bacteria were isolated by agar plating technique, from
rhizosphere of A. viridis, which was grown on naturally
arsenic contaminated site. Isolated bacteria were further
allowed to grow on different concentration of arsenate
As(V) that were 10 M, 100 M, 1 mM on Tryptone Yeast
extract Glucose (TYEG) agar plate. Bacterial colonies
grown on 1 mM plate was considered as more tolerant,
from which one isolate was selected randomly and identified as Bacillus licheniformis by 16SrDNA sequencing. Phylogentic tree (Fig. 1) showing the relationship of B.
licheniformis DAS-1 (accession number KF664027) with

Effects of arsenate [As(V)] and arsenite [As(III)] on

growth of bacteria and MIC
B. licheniformis was grown in TYEG broth amended with 3
mM, 6 mM, 9 mM and 10 mM As(V) and 2 mM, 4 mM, 6
mM, 7 mM As(III) to find out the MIC and growth pattern.
The turbidity of cultures was measured as absorbance
(O.D.) at 600 nm at different time points of growth phase. B.
licheniformis grown in TYEG broth without added arsenic
i.e. control, took 2 h for lag phase then up to 24 h as exponential phase and from 26 h onwards it spent in stationary
phase (Fig. 2A and Fig. 2B). Growth of B. licheniformis is
dependent on concentration of supplied As(V) in medium.
There was gradual decrease in cell growth by increasing
As(V) concentration in medium. About 20% cell growth was
reduced in 3 mM As(V), 40% in 6 mM As(V), 61% in 9 mM
As(V) and 98% in 10 mM As(V). The duration of lag phase
was also gradually increased on increasing As(V) concentration in medium, 10 h of long lag phase was observed in 9
mM As(V) enriched culture. 10 mM was determined as the
MIC since there was no significant growth at this concentration.
The growth pattern of B. licheniformis in arsenite (AsIII)
enriched media has been depicted in Fig. 2B. Cell growth
was gradually reduced on increasing As(III) concentration

Fig. 1. Phylogenetic tree showing relationship of Bacillus licheniformis DAS-1 (KF664027) with the other member of Bacillus
sp. in the NCBI database constructed by neighbour-joining method. Accession numbers of selected sequences are given in
parentheses. Scale bar represents 0.002 substitution per nucleotide position.

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tolerant [1, 32]. Reports on single species having both

[As(V) and As(III)] tolerance capability is rare and very few
[5]. Bacillus licheniformis had tolerated both As(V) and
As(III) at different concentration with MIC for As(V) is
10 mM and for As(III) is 7 mM. This is very significant finding, because here single bacterial species having tendency
to tolerate both forms of arsenic.
The significant inhibition in growth of Bacillus licheniformis
under arsenic stress is showing the toxic effects of As(V)
and As(III). Arsenite (MIC 7 mM) was more toxic than arsenate (MIC 10 mM), which is similar to other reports [23]. It
might be due to arsenite, has very high affinity for protein
thiols so it readily chelated with intracellular proteins and
cause damage to biomolecules of cell [26]. Arsenate act as
substitute of phosphate and inhibit oxidative phosphorylation resulting cell toxicity [17, 46].

Fig. 2. Growth pattern of Bacillus licheniformis in arsenic

containing TYEG broth, over the range of arsenic concentrations (A) Arsenate [As(V)] (3 mM, 6 mM, 9 mM and
10 mM), (B) Arsenite [As(III)] (2 mM, 4 mM, 6 mM and
7 mM). Control cultures with no added arsenic [As(V) or As(III)]
i.e. (0 mM) are shown. Change in OD600 (Absorbance) of culture was measured over 34 h. Error bars indicate the standard
error of the mean of three experiments.

in medium. 24% of reduction was observed at 2 mM As(III),

32% at 4 mM As(III), 55% at 6 mM and 90% at 7 mM As(III)
enrichment. There was gradual increase in duration of lag
phase with increasing As(III) concentration, longest lag
phase i.e. of 12 h was observed in 6 mM As(III) enrichment.
There are various studies of plants utilized for uptake of
metal, metalloid including arsenic [3, 39, 40]. But to utilize
plant associated native bacterial flora for remediation of
arsenic contamination will be a better tool to minimise the
impact of contamination. Generally we find reports on
bacterial species tolerating either As(V) or As(III), like
Paracoccus, Alcaligenes and Pseudomonas, Bacillus were
reported earlier as As(V) tolerant species [4, 12, 47]. Pseudomonas and Corynebacterium were reported as As(III)

http://dx.doi.org/10.4014/kjmb.1401.01004

Potential of uptake/removal of As(V) and its reduction to

As(III)
The uptake/removal potential of Bacillus licheniformis
was dependent on supplied amount of As(V) in the growth
media. The concentration of As(V) left in residual medium
and concentration of As(III) formed in residual medium [culture medium was previously enriched with As(V)] and concentration of As estimated in biomass, at different time of
growth phase has been depicted in Fig. 3A-C. Hence figures depicting the uptake/removal potential of As(V) and
efficiency of reduction of As(V) to As(III). In 3 mM of As(V)
enriched media, 100% As(V) uptake/removal potential was
observed, as no As(V) was found in residual media at the
end of growth experiment. There was gradual decrease in
As(V) uptake/removal at 6 and 9 mM of As(V) enriched
medium, the uptake/removal percentage were 76 and 35
respectively. As(V)/Arsenate (AsO43) is similar to phosphate (PO43) hence enter into cell through phosphate
transport membrane system [38, 46]. Nearly every organism prokaryotes or eukaryotes have natural defence mechanism for arsenic detoxification mostly involved transportation,
oxidation/reduction, extrusion and immobilisation [5, 6, 43].
Most of the microorganism in culture shows at least one
type of As-transforming mechanism, since As(V) is the predominant in oxidized environment [34] and microbial reduction of As(V) to As(III) is an important factor to increase the
mobility and bioavailability of As [20, 27]. In present study
we found the reduction of uptaken As(V) to As(III) and
extrusion of reduced As(III) in growth media. Efficiency of

Uptake/removal and Reduction of As Species by B. licheniformis

reduction of uptaken As(V) into As(III) was also dependent

on concentration of As(V) supplied in media. 62% of
uptaken As(V) was reduced to As(III) with 0.06 mM per h
reduction rate in 9 mM As(V) enriched media, whereas the
56% of uptaken As(V) reduced to As(III) with reduction rate
of 0.086 mM per h, in 6 mM As(V) enrichment and 42% of
reduction with 0.046 mM per h reduction rate was estimated in 3 mM As(V)-enriched media. It was observed that
Bacillus licheniformis has uptaken As(V) first and then
reduced to As(III) which was extruded in the growth media,
as the concentration of As(V) was gradually decreased and
concentration of As(III) was gradually increased in residual
media as presented in Fig. 3A-C. It might be explained in
the manner that As(V) first enters into bacterial cell with the
help of phosphate transporter system, then reduced to
As(III) inside the cell with the help of (arsC) gene product
arsenate reductase [30]. As(III) accumulated in cell then

243

extruded by (arsB) gene product i.e. an antiporter protein

channel [29]. Fig. 3D represents the correlation between
As(V) uptake and As(III) formation, with r = 0.98 and p <
0.001, showing the positive and very significant correlation.
In biomass, up to 1 mM arsenic has been detected,
which is very less as comparison to As(V) supplied. There
is possibility of volatilization of very less amount of arsenic
of total supply. Bioaccumulation of arsenic was also
reported in few species [16].
Uptake/removal potential of As(III)
B. licheniformis has tolerated upto 6 mM As(III) (MIC
7 mM), which might be considered as hypertolerant for
As(III). Because in maximum reports only 1-5 mM As(III)
tolerant rhizospheric bacterial species had been isolated
from natural arsenic contaminated site [21, 25]. To check
the percentage uptake/removal of arsenite [As(III)], concen-

Fig. 3. X-axis represents the concentration of As(V) left in residual media at the same time concentration of As(III) [uptaken
As(V) reduced to As(III)] formed in media along with the concentration of total arsenic (As) accumulated by bacteria [As
in biomass], at different time point (Y-axis) of growth phase. (A) 3 mM As(V) enriched media. (B) 6 mM As(V) enriched media.
(C) 9 mM As(V) enriched media. All values are mean of three replicates and standard errors (SE) are presented as error bars ().
(D) correlation between As(V) uptake and As(III) formation, with r = 0.98 and p < 0.001.

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tration of As(III) left in residual media and concentration of

arsenic in biomass was estimated at different time point of
growth phase as represented in Fig. 4A-C As(III) uptake/
removal potential of Bacillus licheniformis was 100% at
lower concentration of supplied As(III) in media (2 mM),
whereas uptake/removal potential was 75% at 4 mM and
40% at 6 mM of supplied As(III). As(III)/Arsenite (AsO2)
occurs in its hydroxide form as As(OH)3 at neutral pH,
which is an inorganic equivalent of non-ionized glycerol,
hence As(III) uses glycerol membrane transport system to
move across the cell [31, 42]. There was no biotransformation of As(III) in other form. The accumulation of arsenic in
biomass was up to 2 mM, which was higher as compared
to As(V) enriched medium. Very little amount of arsenic
was unaccounted, which might be volatilised or converted
to other organic forms. Since there is no such report
regarding As(III) uptake by bacteria, hence this is new and
significant finding of present study.

Fig. 4. X-axis represents the concentration of As(III) left in

residual media, depicting the concentration of total arsenic
uptaken/removed and accumulated by bacteria [As in biomass] at different time point (Y-axis) of growth phase.
(A) 2 mM As(III) enriched media. (B) 4 mM As(III) enriched
media. (C) 6 mM As(III) enriched media. Concentration of
As(III) gradually decreased in residual media showing removal
of As(III) from the medium. All values are mean of three replicates and standard errors (SE) are presented as error bars ().

http://dx.doi.org/10.4014/kjmb.1401.01004

Effects of pH on growth and uptake/removal of Bacillus

licheniformis in arsenate As(V) and arsenite As(III)
stress
The pH are highly significant variables controlling arsenic
speciation [14]. Availability of arsenic species in growth
medium depend on pH. At around neutral pH, As(V) found
in ionic form as Arsenate (AsO43) and As(III) found in non
ionic condition as As(OH)3. Hence around neutral pH, availability of As(V) and As(III) is higher for uptake by bacteria
[44]. It was observed in this study, that pH of TYEG broth or
control medium (without added arsenic) was 7.2, which
was increased to 7.8 on adding 9 mM As(V) and to 8.7 on
adding 6 mM As(III). Hence pH 6 and pH 9 was taken as
acidic and basic pH change of As(V) stress and pH 6 and
pH 10 for As(III) stress. In present study, growth pattern of
Bacillus licheniformis in As(V) stressed medium, was
affected by variation in pH. In Fig. 5A it was observed that
both acidic and basic pH change of As(V) stressed
medium, had favoured the growth of bacteria. It might be
due to significant decrease in uptake of As(V), in changed
acidic and basic pH condition of As(V) supplied medium as
shown in Fig. 5B. Students t-test between pair data of
uptake at pH 7.8 and at pH 6, with p = 0.0413 and between
uptake at pH 7.8 and pH 9 with p = 0.038. Here change of
uptake in both the case is very significant (p < 0.05). Hence
it is proved that, availability of As(V) in ionic form, is less at
acidic and basic pH, which reduced the uptake. Because

Uptake/removal and Reduction of As Species by B. licheniformis

Fig. 5. Growth pattern and uptake of Bacillus licheniformis

affected by changed pH condition of arsenic enriched
media. (A) 9 mM As(V) enriched media. Three control (no
added arsenate) with pH 7.2, 6 and 9, medium enriched with
9 mM As(V) has pH 7.8 which, was changed to [As(V) pH 6]
and [As(V) pH 9]. Changed pH of As(V) stress favoured the
growth. (B) X-axis represents the concentration of As(V)
uptaken, from 9 mM As(V) enriched media with pH 7.8, 6 and
9. Uptake reduced on changing pH of As(V) stress.

As(V) only can be uptaken via phosphate transporters in

ionic form (AsO43). It was also observed in Fig. 5A that
acidic pH (pH 6) of As(V) stressed medium, was slightly
more favourable for growth than basic pH (pH 9). It was
due to, at alkaline pH, soluble As(V) was more available
than at acidic pH [28]. Since more the availability of soluble
As(V), more the uptake of As(V) by bacterial cell, hence
more the toxic effect resulting less number of cell in growth
phase. There are few reports regarding effect of pH on
arsenic uptake [19] one was reported in mutant strain of S.
faecalis in which the uptake of As(V) was maximal at neutral pH and was declined at higher pH above 7.
Growth of bacteria in As(III) stress was also increased on
exposing to acidic and basic pH change Fig. 6A. There was
also significant effect of pH on As(III) uptake as shown in
Fig. 6B that, change in pH significantly (p < 0.05) reduced

245

Fig. 6. (A) 6 mM As(III) enriched media. Three control (no

added arsenite) with pH 7.2, 6 and 10, medium enriched with
6 mM As (III) has pH 8.7 which was changed to [As(III) pH 6]
and [As(III) pH 10]. Change in pH of As(III) stress reduced the
As(III) toxicity and hence enhanced the growth. (B) X-axis represents the concentration of As(III) uptaken/removed, from
6 mM As(III) enriched media with pH 8.7, 6 and 10. As(III)
uptake reduced in changed pH condition of As(III) stress. All
values are mean of three replicates and standard errors(SE)
are presented as error bars ().

the uptake with p = 0.038 in acidic pH and p = 0.035 in

basic pH. It was due to less availability of As(III) in the form
of non-ionic [As(OH)3], for uptake via glycerol transport at
changed pH condition than at normal pH. There was also
one more interesting observation, that arsenic was found
as favourable factor for growth of Bacillus licheniformis, in
acidic and basic pH condition of TYEG medium, as found in
Fig. 5A and 6A Growth in acidic and basic pH condition of
control medium was very less as compared to medium with
arsenic supplied at same pH.
Variation in total protein content of bacterial cell in different stress
Under different growth condition/stress the cell size and

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Tripti et al.

tively) variation was observed in amount of protein content

of bacterial cell in different stress condition. It might be
explained as due to stress the cell division was inhibited but
there was enlargement of cell size/volume, hence the level
of protein per cell was increased The highest amount of
protein was determined in As(V) (9 mM) stress condition
and lowest amount in control (no added arsenic) as shown
in Fig. 7A. Protein content in As(V) stress with changed
acidic and basic pH condition was significantly lower
(p = 0.0032 for acidic change and p = 0.0094 for basic
change) than only As(V) stress. This result suggests the
change in pH of arsenic stress, modifies/lessen the level of
arsenic stress [13]. Similar pattern of variation in protein
content was also obtained in As(III) stress in Fig. 7B
(p = 0.0153 for acidic change and p = 0.0204 for basic
change of As(III) stress) explaining the same phenomena.

Conclusion

Fig. 7. Total protein content of bacterial cell in different

stress condition. X-axis represents the amount (g) of protein
per unit absorbance (cell density) at different time point (Y-axis)
of growth phase. All values are mean of three replicates and
standard errors (SE) are presented as error bars (). (A) Different pH of As(V) [9 mM] stress (B) Different pH of As(III) [6
mM] stress.

thus protein content can change to several folds [41].

Hence study of variation in level of total cell protein can
measure the level of stress. Cell protein was extracted from
the four set of culture tubes that were control culture (pH
7.2), As(V) stressed culture (pH 7.8) and As(V) stress
exposed to acidic and alkaline pH (6 and 9) at different time
point of growth phase. Similar set was prepared for As(III),
which contained, control (pH 7.2), As(III) (pH 8.7) and
As(III) with pH (6 and 10). Extracted protein was quantified
and represented in graph in the form of amount (g) of protein per unit absorbance/cell density with time in Fig. 7A for
As(V) stress and in Fig. 7B for As(III) stress. Significant
(p < 0.01 and p < 0.05 for As(V) and As(III) stress, respec-

http://dx.doi.org/10.4014/kjmb.1401.01004

B. licheniformis was isolated from arsenic contaminated

region located at 85 32'E longitude and 25 11'N latitude
on the Earth. Isolated bacteria tolerated both As(V) [MIC 10
mM] as well as As(III) [MIC 7 mM], which is rare to find that
single species tolerating both forms of arsenic. Uptake/
removal potential of B. licheniformis was dependent on
supplied amount of As(V) and As(III) in the growth media
and 100% uptake/removal was determined in lower concentration of As(V) and As(III) (3 mM and 2 mM, respectively). B. licheniformis was also capable of reducing
uptaken As(V) into As(III), and its potential of reduction was
also dependent on concentration of supplied As(V). It was
also capable of accumulating little amount of arsenic in biomass. The potential of uptake, reduction and growth pattern
were also affected by variation in pH of arsenic stress. Variation in pH mitigates the arsenic stress, as different level of
cells protein (showing level of stress) was determined at
different pH of arsenic stress. Hence native bacteria can be
utilized in minimizing the arsenic contamination and its efficiency of uptake/removal can also be modify by changing
pH of system, so it can help to avoid the entry of the arsenic
in human food chain.

Acknowledgments
Author pay their sincere gratitude to University Grants
commission, New Delhi, India [F.No.33-169/2007(SR)] and

Uptake/removal and Reduction of As Species by B. licheniformis

Council of Scientific and Industrial Research, New Delhi,

India [F.No.38(1165)/07/EMR II], for providing financial
assistance for the purchase of instrument utilized for this
work. The author would like to thank the anonymous
reviewers for the evaluation of this manuscript.

References
1. Anderson LCD, Bruland KW. 1991. Biogeochemistry of arsenic in natural waters: importance of methylated species. Environ. Sci. Technol. 25: 420-427.
2. APHA, AWWA, WEF. 2005. In Eaton AD, Clesceri LS, Rice
EW, Greenburg AE (eds.), Standards methods for the examination of water and waste water. American public health
association, American water works association, Water Environment Federation Joint Publication USA.
3. Azaizeh H, Salhani N, Sabeswary Z, Shardendu S, Emons H.
2006. Phytoremediation of Selenium using sub-surface flow
constructed Wetlands. Int. J. Phytorem. 8: 187-198.
4. Bachate SP, Cavalca L, Andreoni V. 2009. Arsenic resistant
bacteria isolated from the agricultural soil of Bangladesh and
characterization of arsenic reducing strain. J. Appl. Microbiol.
107: 145-156.
5. Banerjee S, Santra SC. 2010. Studies on the role of aerobic
soil bacteria in arsenic transformation and mobilization. Int. Q.
J. Life. Sci. 2: 587-594.
6. Bhattacharjee H, Rosen BP. 2007. Arsenic metabolism in prokaryotic and eukaryotic microbes. In Nies DHS, Simonies
(ed). Mol. Microbiol. Heavy Met. 6: 371-406.
7. Bradford M. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254.
8. Castaldini M, Turrini A, Sbrana C, Benedette A, Marchionni M,
Mocali S, et al. 2005. Imapct of Bt corn on rhizopheric and soil
eubacterial communities and on beneficial Mycorrhizal symbiosis in experimental microsome. Appl. Environ. Microbiol. 71:
6719.
9. Cherian T, Narayana B. 2005. A new spetrophotometric
method for determination of arsenic in environmental and biological samples. Anal. Lett. 38: 2207-2216.
10. Chakraborti D, Sengupta MK, Rahman MM, Ahamed S. 2004.
Groundwater arsenic contamination and its health effects in
the Ganga-Meghna-Brahmaputra plain. J. Environ. Monit. 6:
74N-83N.
11. Chowdhury R, Sen AK, Karak P, Chatterjee R, Giri AK,
Chaudhuri K. 2009. Isolation and characterization of arsenic
resistant bacterium from a bore well in west Bengal India.
Ann. Microbiol. 59: 253-258.
12. Clausen CA. 2000. Isolating metal-tolerant bacteria capable of
removing copper chromium and arsenic from treated wood.
Waste Managem. 18: 264-268.
13. Ferjani A, Mustardy L, Sulpice R, Marim K, Suzuki I, Hage-

247

mann M. et al. 2003. Glucosylglycerol a compatible solute,

sustain cell division under salt stress. Plant Physiol. 131:
1628-1637.
14. Fitz WJ, Wenzel WL. 2002. Arsenic transformation in the soilrhizosphere-plant system: fundamentals and potential application to phytoremediation. J. Biotechnol. 99: 259-278.
15. Frankerberger WT Jr (ed.) 2002. Environmental chemistry of
arsenic. Marcel Dekker, Inc., New York.
16. Ghodsi H, Hoodaji M, Tahmourespour A, Gheisari MM. 2011.
Investigation of bioremediation of arsenic by bacteria isolated
from contaminated soil. Afr. J. Microbiol. Res. 5: 5889-5895.
17. Goyer RA, Clarkson TW. 2001. Toxic effects of metals. Casarett and Doulls Toxicology: The basis of poison, 6th Ed. Mc
Graw Hill.
18. Grifoni A, Bazzicalupo MC, Serio CD, Fancelli S, Fani R.
1995. Identification of Azospirillum strain by restriction fragment length polymorphism of 16S-rDNA and of the histidine
operon. FEMS Microbiol. Lett. 127: 85-91.
19. Harold MF, Harold LR, Abrams A. 1965. A mutant of Streptococcus faecalis defective in phosphate uptake. J. Biol. Chem.
240: 3145-3153.
20. Harvey CF, Swartz CH, Badruzzaman ABM, Keon-Blute N,
Yu W, Ashraf AM, et al. 2002. Arsenic mobility and groundwater extraction in Bangladesh. Science 298: 1602-1606.
21. Huang A, Teplitski M, Rathinasabathi B, Ma l. 2010. Charaterization of arsenic resistant bacteria from the rhizosphere of
arsenic hyperaccumulator Pteris vittata. Can. J. Microbiol. 56:
236-246.
22. Islam FS, Gault AG, Boothman C, Polya DA, Charnock JM,
Chatterjee D, et al. 2004. Role of metal-reducingbacteria in
arsenic release from Bengal delta sediments. Nature 430: 6871.
23. Kaltreider RC, Davis AM, Lariviere JP, Halminton JW. 2001.
Arsenic alters the function of glucocorticoids receptor as a
transcription factor. Environ. Health Perspect. 109: 245-251.
24. Kolker A, Koeppen RP. 1998. Arsenic variation in pyrite in
coal: Proceeding of the fifteenth international Pittsburg Coal
Conference, 9 p.
25. Liao VH-C, Chu V-J, Su Y-C, Hsiao S-Y, Wei C-C, Liu C-W, et
al. 2011. Arsenite oxidising and arsenate reducing bacteria
associated with arsenic rich ground water in Taiwan. J. Contam. Hydrol. 123: 20-29.
26. Liu SX, Athar M, Lippai I, Waldren C. 2001. Induction of oxyradical by arsenic : implication for mechanism of genotoxicity.
Proc. Natl. Acad. Sci. 98: 1643-1648.
27. Macur RE, Wheeler JT, McDermott TR, Inskeep WP, 2001.
Microbial populations associated with the reduction and
enhanced mobilization of arsenic in mine tailings. Environ.
Sci. Technol. 35: 3676-3682.
28. Masscheleyn PH, Delaune RD, Jr Patrick WH. 1991. Effects
of redox potential and pH on arsenic speciation and solubility
in the contaminated soil. Environ. Sci. Technol. 25: 14141429.

September 2014 | Vol. 42 | No. 3

248

Tripti et al.

29. Meng YL, Liu Z, Rosen BP. 2004. AsIII and SbIII uptake by
GIpF and efflux by Ars B in Escherichia coli. J. Biol. Chem.
279: 18334-18341.
30. Mukhopadhyay R, Rosen BP. 2002. Arsenate reductase in
prokaryotes and eukaryotes. Environ. Health Perspect. 110:
745-748.
31. Mukhopadhyay R, Rosen BP, Phung LT, Silver S. 2002.
Microbial arsenic: from geocycle to gene and enzymes.
FEMS. Microbiol. Rev. 26: 311-325.
32. Nagvenkar GS, Ramaiah N. 2010. Arsenite tolerance and biotransformation potential in Estuarine Bacteria. Ecotoxicology
19: 604-613.
33. Oh S, Rheem S, Sim J, Kim S. 1995. Optimising condition for
the growth of lactobacillus casei YIT 9018 in trypton-yeast
extract-glucose medium by using response surface methodology. Appl. Environ. Microbiol. 61: 3809-3814.
34. Oremland RS, Stolz JF. 2003. The ecology of arsenic. Science 300: 939-943.
35. Prescott H. 2002. Laboratory exercise in microbiology. The
McGraw-Hill.
36. Rosen BP, Liu Z. 2009.Transport pathway for arsenic and
selenium: A minireview. Environ Int. 35: 512-515.
37. Rosen BP. 1999. Families of arsenic transporters. Trends.
Microbiol. 7: 207-212.
38. Sanders OI, Rensing C, Kuroda M, Mitra B, Rosen BP. 1997.
Antimonite is accumulated by the glycerol facilitator GlpF in
Escherichia coli. J. Bacteriol. 179: 3365-3367.
39. Shardendu S, Sayantan D, Sharma D, Irfan S. 2012. Luxary
uptake and removal of phosphorus from water column by representative aquatic palnts and its implication for wet land Man-

http://dx.doi.org/10.4014/kjmb.1401.01004

agement. ISRN Soil Sci. 2012: 1-9.

40. Shardendu, Salhani N, Boulyga SF, Stengel E. 2003. Phytoremediation of Selenium by two halophyte species in sub-surface flow constructed wetland. Chemosphere 50: 967-973.
41. Simon M, Azam F. 1989. Protein content and protein synthesis rates of planktonic marine bacteria. Marine Ecol. Prog.
Ser. 51: 145-156.
42. Solis R. 2004. Experimental and theoretical characterization
of arsenite in water: insight into the coordination environment
of As-O. Inorg. Chem. 43: 2954-2959.
43. Tsai SL, Singh S, Chen W. 2009. Arsenic metabolism by
microbes in nature and the impact on arsenic remediation.
Curr. Opin. Biotechnol. 20: 659-667.
44. Tu S, Ma LQ. 2003. Interactive effect of pH, arsenic and phosphorus on uptake of As and P and growth of the arsenic
hyperaccumulator Pteris vittate L. under hydroponics condition. Environ. Exp. Bot. 50: 243-251.
45. Vijayalakshmi S, Usha V, Rather RA. 2011. In vitro responses
of various bacteria to cadmium chloride: growth and biochemical analysis. Asian J. Exp. Biol. 2: 12-17.
46. Wolfe-Simon F, Switzer Blum J, Kulp TR, Gorden GW, Hoeft
SE, Pett-Ridge J, et al. 2011. A bacterium that can grow by
using arsenic instead of phosphorus. Science 332: 11631166.
47. Xiong J, Wang W, Fan H, Cai L, Wang G. 2006. Arsenic resistance bacteria in mining wastes from shangrao coal mine of
China. pp.535-540. In Lyon WG, Starrett SK, Hong J, Cambridge MA (eds.). Environment Science and Technology (I),
Academic Science Press, USA.