Journal of Hazardous Materials 262 (2013) 1039–1047
Contents lists available at ScienceDirect
Journal of Hazardous Materials
journal homepage: www.elsevier.com/locate/jhazmat
Influence of inoculation of arsenic-resistant Staphylococcus arlettae on growth
and arsenic uptake in Brassica juncea (L.) Czern. Var. R-46
Shubhi Srivastava a , Praveen C. Verma b , Vasvi Chaudhry c , Namrata Singh a , P.C. Abhilash d ,
Kalpana V. Kumar e , Neeta Sharma f , Nandita Singh a,∗
a
Plant Ecology and Environment Science Division, National Botanical Research Institute, Rana Pratap Marg, Lucknow 226001, UP, India
Genetics and Molecular Biology Division, National Botanical Research Institute, Rana Pratap Marg, Lucknow 226001, UP, India
c
Plant Microbe Interaction Division, National Botanical Research Institute, Rana Pratap Marg, Lucknow 226001, UP, India
d
Institute of Environment & Sustainable Development (IESD), Banaras Hindu University, Varanasi 221005, India
e
Agronomy and Soil Sciences Division, Central Institute of Medicinal and Aromatic Plants, Lucknow 226015, UP, India
f
Plant Pathology Laboratory, Department of Botany, Lucknow University, Lucknow, UP, India
b
h i g h l i g h t s
Arsenic tolerant bacteria isolated from arsenic contaminated rhizosphere of paddy.
The bacteria shows plant growth promoting ability.
It enhanced the arsenic uptake in roots of Brassica juncea.
It helps in phytostabilization of arsenic.
a r t i c l e
i n f o
Article history:
Received 13 December 2011
Received in revised form 30 July 2012
Accepted 9 August 2012
Available online 17 August 2012
Keywords:
Arsenic
Staphylococcus arlettae
Plant growth promoting bacteria
Brassica juncea
Phytostabilization
a b s t r a c t
An arsenic hypertolerant bacterium was isolated from arsenic contaminated site of West Bengal, India.
The bacteria was identified as Staphylococcus arlettae strain NBRIEAG-6, based on 16S rDNA analysis.
S. arlettae was able to remove arsenic from liquid media and possesses arsC gene, gene responsible
for arsenate reductase activity. The biochemical profiling of the isolated strain showed that it had the
capacity of producing indole acetic acid (IAA), siderophores and 1-aminocyclopropane-1-carboxylic acid
(ACC) deaminase. Furthermore, an experiment was conducted to test the effect of S. arlettae inoculation on concurrent plant growth promotion and arsenic uptake in Indian mustard plant [Brassica juncea
(L.) Czern. Var. R-46] when grown in arsenic spiked (5, 10 and 15 mg kg−1 ) soil. The microbial inoculation significantly (p < 0.05) increased biomass, protein, chlorophyll and carotenoids contents in test
plant. Moreover, as compared to the non-inoculated control, the As concentration in shoot and root of
inoculated plants were increased from 3.73 to 34.16% and 87.35 to 99.93%, respectively. The experimental results show that the plant growth promoting bacteria NBRIEAG-6 has the ability to help B. juncea
to accumulate As maximally in plant root, and therefore it can be accounted as a new bacteria for As
phytostabilization.
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
Soil polluted with arsenic (As) represents an important environmental problem due to the toxic effects of this metalloid, and
its accumulation through the food chain which poses long term
risks to human health [1]. During the last few dacades, As contamination has emerged as a serious environmental issue due to
∗ Corresponding author at: Eco-auditing Group, National Botanical Research Institute (C.S.I.R.), Rana Pratap Marg, Lucknow 226001, India. Tel.: +91 522 2297931;
fax: +91 522 2205847.
E-mail address: [email protected] (N. Singh).
0304-3894/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jhazmat.2012.08.019
its rapid entry into various environmental media through natural
and anthropogenic sources [2–4]. In India, especially in West Bengal, the elevated concentration of As in plants and soil is mainly
due to the irrigation of agricultural soil with As contaminated
groundwater [5]. Although various remediation methods – such as
containment, solidification and stabilization etc. has been proposed
for the decontamination of onsite contaminated soils, all of these
methods require appropriate controls and long-term monitoring to
ensure the behavior of As through soil column [6]. However, phytoremediation, especially phytoextraction is getting popularity as
a low cost and inventive method of remediation. Phytoextraction is
a solar driven technology so that it can be successfully deployed for
the cleaning up of the As contaminated soils. In recent years, it has
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S. Srivastava et al. / Journal of Hazardous Materials 262 (2013) 1039–1047
been reported that some plant species (hyperaccumulators) growing in arsenic contaminated soils have the ability of accumulating
high concentration of arsenic [7–11]. However, the field utilization potential of most of these plants are very less due to their
small biomass and slow growth rate [12]. Another possibility is
in situ arsenic phytostabilization, i.e., using metal tolerant plants
for retaining the metal maximally in roots, thus reducing leaching
and uptake in plant parts, further preventing transfer to food chain
[13–15].
Rhizoremediation, involving both plants and the rhizospheric
microbes, is an efficient bioremediation process for contaminant degradation and/or promoting plant growth in presence of
plant growth promoting bacteria (PGPB). Different metal tolerance
mechanisms have also been discovered in various microbes [16,17]:
exclusion, active removal, biosorption, precipitation or bioaccumulation, both in external and intracellular spaces. Some bacterial
strains are also known to play an important role in the biochemical cycle of As, through its conversion to species with different
solubility, mobility, bioavaiability and toxicity [18,19]. The known
mechanism of arsenic resistance in microorganisms requires the
ars operon and is based on energy dependent efflux of both arsenate [As(V)] and arsenite [As(III)] from the cell [20]. In this operon
the gene arsC is particularly interesting because its product, a cytoplasmic arsenate reductase, catalyzes reduction of less toxic As(V)
to more toxic As(III), which may be transported out of the cell by
arsAB, As chemiosmotic efflux system and by ATPase membrane
system [21].
Rhizospheric bacteria have found to enhance [22], reduce, or
have no effect on the metal uptake [23,24]. For instance, Rajkumar and Freitas [23] reported that the addition of Pseudomonas
jessenii to surface sterilized root of Ricinus communis in autoclaved
soil increased Zn concentrations in shoot tissues compared with
non-inoculated controls. In contrast, Vivas et al. [25] reported that
inoculation with PGPB Brevibacillus sp. reduced Zn uptake in Trifolium repens plants.
Indian mustard plant, Brassica juncea (L.)Czern., is known to
accumulate Zn, Cu, Pb, Cr, Cd and As [26–32]. Despite the potential
of B. juncea to remove many toxic metals from the soil, phytoremediation is yet to become commercially available technology as
contradictory results have been reported regarding the efficiency
of this plant in phytoextraction [30,32–34].
The aim of this work was (i) isolation and characterization of As
resistant bacteria from As contaminated soil, and (ii) the evaluation
of the As phytoremediation potential of B. juncea plants in association with arsenic resistant bacteria when grown in arsenic spiked
soil.
temperature at CSIR-National Botanical Research Institute (NBRI),
Lucknow.
All isolated bacterial strains were screened for arsenic resistance
assay. These strains were grown on nutrient agar (NA) plates containing different concentration of As(V) (using sodium arsenate)
ranging from 0.61 to 366 mM and As(III) (using sodium arsenite)
ranging from 0.4 to 40 mM. All plates were incubated at 37 ◦ C for
24 h. After screening bacterial strain namely, NBRIEAG-6 was found
to be resistant to 366 mM As(V) and 20 mM As(III). This bacterial
strain has been selected for further studies.
NBRIEAG-6 strain was subjected to several morphological, physiological, and biochemical tests for its characterization. The same
are summarized in Table 1. For determination of phosphate solubilization ability, the As tolerant bacterial strains were grown
in modified Pikovskaya’s (PVK) medium [36] supplemented with
0.5% of tricalcium phosphate at 30 ◦ C, for 144 h on the rotary
shaker, at 220 rpm. The culture supernatants were collected by
centrifugation at 8000 rpm for 20 min. Soluble phosphate in the
culture supernatants were estimated according to the method
of Fiske and Subbarow [37]. Indole acetic acid (IAA) production was determined according to the method of Bric et al. [38].
An aliquot of 2 ml supernatant obtained from bacterial cultures
grown in Luria–Bertani’s medium supplemented with tryptophan
(500 ␮g ml−1 ), were mixed with 100 ␮l of 10 mM orthophosphoric acid and 4 ml of Salkowski’s reagent. The absorbance of pink
color developed after 30 min incubation and absorbance was read
at 530 nm. The IAA concentration in cultures was determined
using a calibration curve of pure IAA as a standard following
the linear regression analysis. The ability of the isolate to produce siderophores was checked by growing bacterial strains on
modified chrome azural S (CAS) agar plates as described by
Schwyn and Neilands [39]. Instead of glucose, yeast extract (to
a final concentration of 0.04%) was added. The plates were incubated for 4 days in the dark at 22 ◦ C and the formation of
halos around colonies was noted. For 1-aminocyclopropane-1carboxylic acid (ACC) deaminase determination lysates in four
replicates of bacterial cultures were prepared as described by
Saleh and Glick [40]. The microorganism’s lysates were centrifuged
(10,000 g, 10 min) and 1 ml of supernatant was mixed with 800 ␮l
of 0.56 N HCl and 300 ␮l of 2, 4-dinitrophenylhydrazine (0.2 g
in 100 ml of 2 N HCl). The mixtures were incubated for 30 min
at 30 ◦ C then 2 ml of 2 N NaOH was added. The absorbance was
measured at 540 nm. The ACC deaminase activity of bacterial
strains was evaluated quantitatively by measuring the amount
of ␣-ketobutyrate produced by deamination of ACC. ACC deaminase activity was expressed in ␮mol of ␣-ketobutyrate mg
protein h−1 .
2. Materials and methods
2.2. Identification of bacterial strain NBRIEAG-6
2.1. Bacteria isolation and characterization
Soil samples (non-rhizospheric, alluvial soil) were collected
from village Birnagar (latitude-72◦ , 51 , E, longitude-22◦ , 41 , N),
Nadia district, in the state of West Bengal, India. This is an
As rich area featuring soil As concentrations in average, up to
12.43 mg kg−1 [35]. The soil samples were maintained at 4 ◦ C during
transportation to laboratory, for further processing.
Soil samples were serially diluted (up to 10−6 ) and inoculated
on nutrient agar medium, containing peptic digest of animal tissue
10 g l−1 , beef extract 5 g l−1 , sodium chloride g l−1 , agar 0.5 g l−1 and
adjusted to pH 7.0 (Difco Manual 1953). An evenly isolated colony
was developed along the lines of lateral streaks and eventually a
single cell was then transferred on the nutrient agar surface. Fifty
eight isolates were originally obtained in this manner and all are
being maintained on nutrient agar slants in 25% glycerol at −80 ◦ C
Pure culture of the target bacterial strain was grown over night
in liquid nutrient broth (NB) medium for the isolation of genomic
DNA using a method described by Hiney et al. [41].
CSIR-Institute of Microbial Technology (IMTECH), Chandigarh
India, has identified the selected strain as Staphylococcus sps., which
is deposited at IMTECH with accession no. MTCC10219.
Further identification was carried out through PCR amplification of 16S rDNA. A PCR product of 1465 base pairs 16S rDNA
was sequenced, and data were analyzed as described earlier [42].
Sequence data of NBRIEAG-6 have been deposited in the GenBank,
(accession number is JQ388197). Sequence analysis of the isolate
was compared with 16S rDNA sequences using BLAST search in
the NCBI, GenBank database (http://www.ncbi.nlm.nih.gov). The
isolate NBRIEAG-6 showed closest homology (99%) with Staphylococcus arlettae.
S. Srivastava et al. / Journal of Hazardous Materials 262 (2013) 1039–1047
1041
Table 1
Functional, morphological, biochemical and physiological test characterization of isolated bacterial strain.
Tests
Functional characterisation
IAA [␮g mg−1 l]
ACC [␮M ␣-KB mg−1 h−1 ]
Phosphate solubilization [␮g ml−1 ]
Siderophore production
Ars C operon
Presence on As(V) at 0.61 to 366 mM
Presence on AS(III) at 0.4–40 mM
NCBI GEN bank accession number
16s rDNA analysis
Morphological characterization
Gram staining
Configuration
Surface
Pigment
Opacity
Cell shape
Size (␮m)
Motility
Anaerobic
Spores
Biochemical characterization
Nitrate
Citrate
Arginine
Indole
Gelatin hydrolysis
Starch hydrolysis
Esculine hydrolysis
Catalase test
Oxidase test
Methyl red test
Voges proskauer test
Urea production
√
NBRIEAG-6
41.07 ± 2.14
5.10 ± 0.06
102 ± 1.27
√
√
√
√
JQ388197
Staphylococcus arlettae
+ve
Circular
Moist
Whitish
Opaque
Coccus
0.5 ␮
Nonmotile
√
•
√
√
√
√
√
•
√
√
•
•
•
Tests
NBRIEAG-6
Acid production from:
Sucrose
Rhamnose
Dextrose
Fructose
Lactose
Mannitol
Maltose
Xylose
Inositol
Adonitol
Sorbitol
Raffinose
Growth on McConkey
Physiological characterization
Growth at temperature:
4 ◦C
15 ◦ C
25 ◦ C
30 ◦ C
37 ◦ C
42 ◦ C
55 ◦ C
Growth at pH:
pH 5.2
pH 7.0
pH 8.0
pH 9.0
pH 10.5
Growth on NaCl (%):
2.0
4.0
6.0
8.0
10.0
√
√
√
√
•
•
√
√
•
•
√
√
•
√
√
√
√
√
√
•
•
√
√
√
√
√
√
√
√
√
= positive; • = negative; = weak positive.
2.3. Determination of growth kinetics and arsenic removal
capacity from media by bacterial cells
The bacterial strain NBRIEAG-6, was grown at 37 ◦ C in 300 ml
nutrient broth containing 183 mM As (V), 6 mM As (III) into 500 ml
Erlenmeyer flask. As free media was taken as a control. The flasks
were placed in an orbital shaker at 110 rpm at 32 ◦ C. Culture broth of
10 ml was collected at different time intervals (10, 24, 48 and 72 h)
until it reached stationary phase. Bacterial growth was monitored
by measuring optical density of the 1 ml culture at 600 nm using
spectrophotometer (CARY 50 UV, Australia).
One ml sample of each collection was taken for As quantification.
Cells were harvested by centrifugation (5000 × g for 30 min). The
cell pellets were washed 2–3 times with normal saline, to remove
adsorbed As, dried at 70–80 ◦ C to a constant weight and digested
with concentrated nitric acid using a microwave digestion system
(BERGHOF- speedwave- MSW-3+ ). Samples were brought to a constant volume before analysis and then used in the measurements of
As accumulation. The As concentrations in all of the samples were
analyzed by using inductively coupled plasma mass spectrometry
[ICP-MS] (Agilent-7500).
2.4. Detection of arsC genes
For amplification of arsC genes two primer pairs described by
Joshi et al. [18] were used. The primer sequence for arsC amplified
region were arsC F 5 -AAC AGT TGC CGC AGC ATT CT-3 , arsC R 5 TGC GCT CCA GCT CAC GCTT -3 . These primers amplified a fragment
of 350 base pairs of the arsC sequence. The PCR conditions were as
follows: initial denaturation step at 94 ◦ C for 3 min, then 30 cycles
of 94 ◦ C for 30 s, 55 ◦ C (for primer amlt-42-f and amlt-376-r) 72 ◦ C
for 1 min with a final extension of 72 ◦ C for 5 min. Negative controls included a deionizer water reagent control. The presence of
arsC gene in a PCR product of a positive control was confirmed by
sequencing.
2.5. Evaluation of growth and arsenic uptake by B. juncea in
presence of As-resistant bacteria NBRIEAG-6
2.5.1. Physico-chemical properties of the soil
The Garden soil (GS) was collected from NBRI, Lucknow (India).
Soil was sterilized by autoclaving at 121 ◦ C at 15 psi pressure for
1 h and then supplemented with 5, 10 and 15 mg kg−1 As using
sodium arsenate salt. After 5 days of incubation, physico-chemical
analysis of treated garden soil was done by procedures described
by Kalra and Maynard [43]. All the chemicals used were of analytically reagent (AR) grade. Samples were sieved (2 mm) and
stored at 4 ◦ C. Enzyme activities in soil samples were assayed
using fresh moist soil samples from pots. Dehydrogenase activity (DHA) was assayed following the method of Pepper et al. [44]
through the reduction of 2, 3, 5 - triphenyl tetrazolium chloride (TTC) and expressed in ␮g triphenyl formazan g−1 soil h−1 .
Alkaline phosphatase activity was estimated by the method of
Eivazi and Tabatabai [45]. The microbial biomass carbon was determined using the chloroform-fumigation–extraction method Vance
et al. [46] and pH was measured in the aqueous extracts of soil:
distilled water of 1: 2.5 (w/v dry weight basis) using pH meter
(Orion meter) at 25 ◦ C. Total organic carbon was analyzed by using
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S. Srivastava et al. / Journal of Hazardous Materials 262 (2013) 1039–1047
Walkley and Black [47] method. Available phosphorus was analyzed by the method of Olsen et al. [48].
0.9
2.5.2. Experimental setup
A greenhouse experiment was conducted to evaluate the effect
of bacterial isolates on plant growth and As uptake by B. juncea
(L.) Czern.Var. R-46. The seeds of B. juncea were surface sterilized
by soaking for 5 min in 0.1% mercuric chloride solution and then
thoroughly rinsing with sterile distilled water. The sterilized seeds
were sown in sterilized GS. The two weeks old seedlings were transferred in plastic pots filled with 750 g sterilized soil supplemented
with 5, 10 and 15 mg kg−1 As using sodium arsenate salt. In this
experiment the sterilized soil was taken for assessing the effect of
this particular strain without the interference of native bacterial
strain. The experimental design was a randomized complete block
design, with three replicates for each treatment. Three seedlings
were transplanted in each pot.
For inoculation, the selected As hypertolerant bacterial strain
NBRIEAG-6 was grown overnight in 100 ml Erlenmeyer flasks containing 50 ml of sterilized Nutrient broth on a shaker at 150 rpm
at 30 ◦ C. Cells in the exponential phase were collected by centrifugation at 8000 g for 25 min at 4 ◦ C, washed with sterile distilled
water, and centrifuged. Bacterial inoculums were prepared for all
treatments by resuspending pelleted cells in sterile distilled water
to get an inoculum density of approximately 8.1 × 108 CFU ml−1 ,
giving an absorbance of 0.5 at 600 nm.
After one week, the rhizospheres of the seedlings were inoculated with 50 ml pot−1 bacterial suspension. Fifty ml of nutrient
broth was also added to the rhizosphere of control treatment pots.
The pots were kept under greenhouse conditions [18 ◦ C (night) and
24 ◦ C (day), 80% relative humidity, 11 h photoperiod]. The moisture
was maintained as 60% by covering the pots with plastic hood. The
plants were harvested after 30 days of transplanting and growth
parameters like shoot length, root length, fresh and dry weight
were recorded. Total chlorophyll and carotenoids content of B.
juncea leaves were measured in acetone extract by the method of
Machalachlan and Zalik [49]. The protein content of supernatant
was estimated by the method of Bradford [50] using bovine serum
albumin as standard.
0.7
2.5.3. Arsenic estimation in soil and plant tissue
After harvesting, plants roots and shoots were separated and
washed extensively with distilled water. The roots were kept in
EDTA (20 mM) solution for 15 min [51] to remove the adhering
metal on the root surface. Samples were oven dried at 75 ◦ C till constant weight was achieved and powdered. The powdered soil and
plant material (0.1 g DW) were digested in BURGHOF-speedwaveMSW-3+ microwave digestion unit with 5 ml of 60% nitric acid and
1 ml of 40% hydrofluoric acid and filtered through Whatman filter
paper no. 44. As in the digested samples was determined by inductively coupled plasma mass spectrometry [ICP-MS] (Agilent-7500
cx).
A Translocation factor (TF) was calculated by using the following
formulae:
TF (root to shoot) = mean accumulation of metal by shoot
part/mean accumulation of metal by root part.
2.5.3.1. Quality control and quality assurance. The standard reference material of arsenic (E-Merck, Germany) was used for each
analytical batch. Analytical data quality was ensured with repeated
analysis of quality control samples and the results were within
92–98% limit of the certified values. Standard AA03N-3 (Accustandard, USA) was used as a matrix reference material which
was spiked with known concentration (0–50 ␮g L−1 As) of standard reference material, and the recovery of total As were within
85.3–89.5%.
0.8
O.D. (600nm)
0.6
0.5
0.4
0.3
0.2
0.1
0
0h
12 h
NBRIEAG-6
24 h
NBRIEAG-6 +As(V)
48 h
72 h
NBRIEAG-6 +As(III)
Fig. 1. Growth of bacterial strains at 183 mM As V and 6 mM As(III) at different time
intervals.
3. Result and discussion
3.1. Isolation and identification of arsenic resistant bacteria
Arsenic has been reported to exert toxic effects on microorganisms through various mechanisms [52]. In the initial screening, in
arsenic amended nutrient agar media, the isolated strain NBRIEAG6 showed resistance to 366 mM As(V) and 40 mM As(III). But the
best growth was found to be at 244 mM As(V) and 20 mM As(III).
Microbial resistance to As is widespread in nature [53–55]. As levels tested in this study are much higher compared to those found in
contaminated soil. Jackson et al. [55] reported As resistant levels of
400 mM and 10 mM of As(V) and As(III), respectively. Studies conducted by Escalante et al. [56] reported As resistant isolates capable
of tolerating As(V) above 1000 mM and As(III) concentration to
40 mM. Resistance to concentration of As(V) higher than 100 mM
is considered as very high by Jackson et al. [57]. The tolerance
level reaching 300–500 mM of As(V) could be considered as hypertolerant according to Drewniak et al. [58]. Such high resistance
to As(V) has also been considered as biotechnologically important
bacterium Corynebacterium glutamicum [59] and Bacillus sp. Strain
DJ-1 [18].
The morphological, biochemical and physiological descriptions
of the isolated strain NBRIEAG-6 are summarized in Table 1. The
selected strain colonies were small and whitish in color. The strain
was characterized as gram positive, coccus, with cell size 0.5 ␮.
Based on the results of the IMTECH, this bacteria was identified as
Staphylococcus sps. with accession no. MTCC10219. Further identification of NBRIEAG-6 was carried out based on 16S rDNA gene
sequence analysis. A PCR product of 1465 base pairs 16S rDNA
gene was sequenced, and data were analyzed as described earlier [42]. Sequence analysis of the isolate was compared with 16S
rDNA sequences using BLAST search in the NCBI, GenBank database,
showing closest 99% homology with S. arlettae. Sequence data of
NBRIEAG-6 has been deposited in the GenBank, with accession
number JQ388197.
3.2. Growth kinetics and arsenic removal capacity of bacterial
cells from liquid media
The growth rate constant (k) for the log phase of growth was
determined by plotting the log10 of the optical density against
time. Data in Fig. 1 show, that NBRIEAG-6 has higher growth
rate in As(V) containing media as compared to As(III) containing
medium, although it was around 6 times less when no As was
S. Srivastava et al. / Journal of Hazardous Materials 262 (2013) 1039–1047
1043
Fig. 2. Arsenic uptake in inside the bacterial strain at (a) 183 mM AsV and (b) 6 mM AsIII on different time intervals.
added in the media. Results expressing the capability of NBRIEAG6 to uptake As(V) and As(III) are given in Fig. 2. The accumulation
of As(V) and As(III) by bacterial strain increased with increasing
time period in h. These results were in accordance with Joshi et al.
[18]. The higher sorption was achieved after 48 h and 72 h of incubation period. Further incubation did not improve the growth and
extent of biosorption. The study suggests that the isolate NBRIEAG6 appears to be efficient for removing both As(III) and As(V) from
liquid medium.
3.3. arsC detection in isolated bacterial strain
The ars system is a widely studied As resistance mechanism,
where arsC gene codifies a soluble enzyme, arsenate reductase,
which catalyzes As(V) reduction to As(III) [60]. The presence of arsC
gene was confirmed in isolated bacterial strain NBRIEAG-6 (Table 1
and Fig. 3). However, researchers have also reported that amplification of the ars genes and unknown chromosomal ars areas during
a PCR is influenced by many factors, such as the type of primer
used, conformational variation in the extracted DNA, thermal cyclic
conditions and the composition of buffers or agents [61,62]. Here
we had used specific PCR primers based on the sequence of arsC
as reported earlier by Joshi et al. [18]. Huang et al. [21] have reasoned that As(V) is reduced to As(III) by arsC and detoxification
processes based on As(V) reductase activity may be significant in
dissemination of As pollution. Recently, many studies have focused
on the detection of ars genes to correlate their presence with the As
resistance phenotype of bacterial isolates or to use these genes as
potential molecular biomarkers of As contamination [57,63]. These
results have also been supported by Drewniak et al. [58] and Joshi
et al. [18] in their studies.
3.4. Plant growth promoting (PGP) characteristics of the isolated
strain
Certain metal resistant bacteria have been shown to improve
host plant growth and mitigate toxic effects of metal on the
plants [23,64]. To identify potential PGPR, the As-resistant isolate
NBRIEAG-8, was qualitatively screened for its ability, (i) to produce IAA in medium supplemented with l-tryptophan; (ii) to utilize
ACC as the sole N source; (iii) to solubilize phosphorus, and (iv)
to secrete siderophore into the growth medium. Table 1 shows
that the bacteria possessed all PGP traits. Maximum IAA production
(∼41 ␮g mg−1 ) was observed by S. arlettae (NBRIEAG-6) after 48 h of
incubation. The results correspond with earlier observations which
indicate induction of IAA in stationary phase culture [64]. Such
findings may have direct practical application, although intrinsic
ability of bacteria to produce IAA in the rhizosphere depends on
the availability of precursors and uptake of microbial IAA by plant
[65].
The solubilization of phosphate (102 ␮g ml−1 ) by S. arlettae
strain (NBRIEAG-6), was achieved after 120 h of incubation. Further incubation had no effect on the extent of solubilization. Final
pH of the growth medium was recorded at regular interval of 24 h to
Fig. 3. Gel photograph of arsC operon.
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S. Srivastava et al. / Journal of Hazardous Materials 262 (2013) 1039–1047
Table 2
Changes in pH, dehydrogenase enzyme, phosphatase enzyme, microbial biomass carbon, total organic carbon and available phosphorus levels in two As treated groups
(noninoculated and inoculated) at three different concentrations. Values are mean ± S.D. Comparison within pairs ns- p > 0.05, *- p < 0.05, **- p < 0.01.
Serial no.
Parameters
1
pH
Control (only As)
NBRIEAG-6
As concentration (mg kg−1 )
5
2
3
4
5
6
10
15
8.9 ± 0.11
7.6 ± 0.45**
9.0 ± 0.10
7.8 ± 0.21**
9.7 ± 0.10
8.4 ± 0.35**
0.07 ± 0.02
0.57 ± 0.11**
0.08 ± 0.05
0.54 ± 0.19**
0.08 ± 0.05
0.54 ± 0.19**
Phosphatase enzyme (␮g g−1 )
Control (only As)
NBRIEAG-6
15.23 ± 6.29
52.23 ± 0.58**
23.19 ± 0.29
53.43 ± 0.58**
33.25 ± 0.31
53.81 ± 0.33**
Microbial biomass carbon (␮g g−1 )
Control (only As)
NBRIEAG-6
21.73 ± 3.43
29.83 ± 2.67 ns
6.93 ± 5.48
35.17 ± 4.53**
10.23 ± 6.29
30.45 ± 4.95**
Total organic carbon (%)
Control (only As)
NBRIEAG-6
0.21 ± 0.08
0.71 ± 0.06**
0.25 ± 0.02
0.67 ± 0.03**
0.29 ± 0.05
0.53 ± 0.05**
Available phosphorous (␮g g−1 )
Control (only As)
NBRIEAG-6
0.43 ± 0.05
0.13 ± 0.05**
0.23 ± 0.01
0.53 ± 0.05**
0.13 ± 0.05
0.36 ± 0.01**
Dehydrogenase enzyme (␮g g−1 )
Control (only As)
NBRIEAG-6
find out if solubilization was accompanied by acid production. The
decrease in pH (data not shown), clearly indicates the production
of organic and inorganic acids which are considered to be responsible for phosphate solubilization [66]. It has been well documented
that solubilization of phosphate in the rhizosphere makes a major
contribution to the growth promoting effect of bacteria on plants
[19,67].
In order to find the PGP activity, the As-resistant strain was
tested for its ability to grow on salt minimal medium with ACC.
Bacterial strain utilizing ACC as a sole source of nitrogen, possess
ACC deaminase which hydrolysis ACC into ␣-KB and ammonia, and enhances the elongation of plant roots [68] and plant
growth under metal stress condition [69]. The bacterial strain,
S. arlettae recorded the high ACC deaminase activity (5.10% ␮M
␣KB mg−1 h−1 ) (Table 1).
The siderophore is another important metabolite released by
the plant growth promoting bacteria that directly alleviates heavy
metal toxicity by increasing the supply of iron to the plant [70]. In
this study, the As resistant isolate showed presence of siderophores
(Table 1). During this process, As(V) can be mobilized from the
solid to aqueous phase, and in some microsites As concentrations
can reach much higher levels than the average value in the examined environment. Thus, the presence of siderophores may also
provide an explanation for the extreme As resistance of bacteria
[71].
3.5. Changes in soil properties
The physico-chemical analysis of treated sterilized garden soil
(zero day data) shows alkaline pH (8.8–9.4), phosphorus 0.1%, nitrogen 1.3% at 5 and 10 mg kg−1 , and 1.2% at 15 mg kg−1 , potassium
content in soil was 1.3% and organic carbon 0.30%. The As concentration was 4.33, 9.86 and 12.78 mg kg−1 at 5, 10 and 15 mg kg−1
treatments, respectively.
Physico-chemical properties of inoculated soil are presented
in Table 2. Except for pH all other tested soil properties (DHA,
phosphatase enzyme, microbial biomass carbon, total organic carbon, and available phosphorus) increased. The reduction in the pH
may be due to the action of siderophores [72]. There was significant increase in microbial biomass carbon, due to the inoculation
of bacteria. The increase in phosphorus in inoculated soil may
have been due to the phosphate solubilizing activity of the isolated bacterial strain. The soil enzymes, viz., DHA and phosphatase
increased significantly after bacterial inoculations. Thus, tested
bacterial strain improved the soil characteristics for the better
growth of B. juncea.
3.6. Influence of PGPB on growth of B. juncea
The B. juncea plants were inoculated with the As resistant
strain to examine the PGP ability of the strain and its effect on
As translocation in shoot and root. The growth of plant (shoot
length, root length, fresh weight and dry weight) and biochemical contents (total chlorophyll, carotenoids and protein) of control
(non-inoculated + As) and NBRIEAG-6 (inoculated + As) at three
concentrations of As (5, 10 and 15 mg kg−1 ) are summarized in
Table 3. Significant increases (p < 0.05) of all parameters were
observed in plants when soil was inoculated with NBRIEAG-6 strain,
compared to the non-inoculated soil (Table 3).
The plant growth promotion by rhizospheric bacteria has been
observed by several authors [73,74] due to the utilization of ACC,
synthesis of phyto-hormone and solubilization of minerals. Studies
have confirmed the potential of ACC utilizing bacteria to promote
the growth of Brassica campestris, B.napus, B. juncea, Lycopersicon
esculentum, Zea mays, Triticum aestivum [19,64,68,75–77]. Jiang
et al. [19] investigated that a heavy metal resistant Burkholderia sp. J62 isolated from heavy metal contaminated soil promoted
plant growth by the synthesis of ACC deaminase. Kumar et al. [78]
isolated a metal tolerant NBRIK28 Enterobacter sp. from fly ash
contaminated soils, which along with its siderophore over producing mutant NBRIK28 SD1, were found capable of stimulating plant
biomass, concurrent production of siderophores, IAA and ACC in
B. juncea grown in fly ash amended soil. NBRIEAG-6 recorded the
higher ACC deaminase activity, which also promoted the long root
(Table 3). The role of ACC deaminase in decreasing ethylene levels by the enzymatic hydrolysis of ACC into ␣-KB and ammonia
has been presented as one of the major mechanisms of PGPB in
promoting root [68] and plant growth. The isolated strain showed
the presence of siderophore producing activity, which enhanced
the plant biomass, chlorophyll and root proliferation. Plants inoculated with siderophore producing bacteria (SPB), take up iron from
siderophores via various mechanisms, such as chelate degradation
S. Srivastava et al. / Journal of Hazardous Materials 262 (2013) 1039–1047
1045
Table 3
Changes in shoot length, root length, fresh weight, dry weight, total chlorophyll, carotenoid and protein levels in two As treated groups (noninoculated and inoculated) at
three different concentrations. Values are mean ± S.D. Comparison within pairs ns- p > 0.05, **- p < 0.01.
Serial no.
As concentration (mg kg−1 )
Parameters
5
1
2
3
4
5
6
7
10
Root length (cm)
Control (only As)
NBRIEAG-6
9 ± 0.5
14.7 ± 2.08**
Root length (cm)
Control (only As)
NBRIEAG-6
5.34 ± 0.58
10.83 ± 0.58**
Fresh wt (g)
Control (only As)
NBRIEAG-6
Dry wt (g)
Control (only As)
NBRIEAG-6
5 ± 0.56
12.5 ± 1.73**
5.17 ± 1.15
8.20 ± 1.57**
0.14 ± 0.04
0.94 ± 0.03**
0.19 ± 0.06
0.85 ± 0.04**
0.11 ± 0.01
0.82 ± 0.01**
0.02 ± 0.01
0.07 ± 0.02**
0.05 ± 0.00
0.12 ± 0.0**
0.04 ± 0.00
0.08 ± 0.00**
Total chlorophyll (mg g−1 )
Control (only As)
NBRIEAG-6
0.57 ± 0.05
0.658 ± 0.08 ns
0.46 ± 0.02
0.52 ± 0.04 ns
0.24 ± 0.00
0.47 ± 0.06**
Carotenoids (mg g−1 )
Control (only As)
NBRIEAG-6
0.17 ± 0.02
0.254 ± 0.04*
0.17 ± 0.00
0.25 ± 0.08*
0.16 ± 0.00
0.24 ± 0.00*
Protein (mg g−1 )
Control (only As)
NBRIEAG-6
45.31 ± 6.24
49.25 ± 4.29 ns
38.24 ± 1.75
47.48 ± 1.93**
31.20 ± 1.38
45.20 ± 2.77**
Control (only As)
High IAA production (41 ␮g mg−1 ) was observed in the tested
strain (Table 1), which is in good agreement with the higher shoot
length, number of leaves and total chlorophyll in B. juncea inoculated with this strain. The results are congruent with findings of
Gravel et al. [75]. According to Glick et al. [82] the IAA produced
by PGPB promoted root growth by directly stimulating plant cell
elongation or cell division.
In addition to IAA production, the phosphate solubilization by
PGPB is also believed to play an important role in plant-bacterial
interactions and plant growth [83]. As(V) in soil tends to reduce the
amount of phosphorus in plants [84,85], which adversely affects the
NBRIEAG-6
Control (only As)
100
a
ns
b
60
**
ns
40
**
20
0
0
5
10
15
10
6
4
**
**
10
5
NBRIEAG-6
80
15
8
6.20 ± 0.52
12.00 ± 0.87**
3.84 ± 0.88
9.5 ± 1.0**
and release of iron [79]. Several examples of a simultaneous growth
promotion and increased Fe-uptake in plants as a result of SPB inoculations have been reported. For instance, Crowley and Kraemer
[80] demonstrated a siderophore mediated iron transport system
in oats and suggested that siderophores produced by rhizosphere
microorganisms could supply iron to oat, which has mechanisms
for using Fe-siderophore complexes under iron-limited conditions.
Sharma et al. [81] assessed the role of the siderophore-producing
Pseudomonas strain GRP3 on iron nutrition of Vigna radiata. After
45 days, the plant showed a significant increase in chlorophyll a
and chlorophyll b contents, when compared with the control.
20
15
**
c
**
**
2
0
5
10
15
Arsenic Concentraon (mg kg -1)
5
14
12
10
8
6
4
2
0
10
15
d
**
5
**
10
**
15
Arsenic Concentraon (mg kg -1)
Fig. 4. As (mg kg−1 ) in (a) shoot, (b) root, (c) soil and (d) translocation factor (root to shoot) in control (only As) and NBRIEAG-6 (As + bacterial inoculated) at three different
concentrations. Values are mean ± S.D. Comparison within pair ns- p > 0.05, **- p < 0.01.
1046
S. Srivastava et al. / Journal of Hazardous Materials 262 (2013) 1039–1047
growth in As sensitive plants. This deficiency can be compensated
by the inorganic phosphate solubilizing ability of selected strain,
reducing the pH of inoculated soil (Table 2).
3.7. Arsenic accumulation in B. juncea
Arsenic in the root and shoot systems increased significantly
(p < 0.05) with increasing concentration of As in the soil (Fig. 4a
and b). Although there was no significant change in the concentration of As in soil (Fig. 4c), this situation changed after inoculation
of As-resistant strain NBRIEAG-6. The bacterial strain stimulated
the As uptake in root and the maximum uptake was observed in
inoculated plants grown in10 mg kg−1 of As. However, the bacterial
inoculation significantly decreased the As uptake in shoot. This can
be further interpret from the translocation factor (TF; uptake from
root to shoot) given in Fig. 4d. The TF was >1 in non-inoculated
plants but it decreased to <0.2 after inoculation with NBRIEAG-6
strain. Burd et al. [86] have also recorded similar observations upon
inoculation with Kluyvera ascorbata under nickel, lead and zinc
stress. In contrast to the present observations, Hasnain and Sabri
[87] have reported decrease in the accumulation of chromium and
growth of Triticum aestivum inoculated with Pseudomonas sp. However, Jiang et al. [19] observed decrease in shoot Cd concentration
of the bacterial (Burkholderia sp. J62) inoculated maize plants compared to the shoot Cd concentration of the non-inoculated maize
plants. In contrast, significant increase was found in root of the
maize plant. Dary et al. [88] found no accumulation of As in yellow
lupines when grown in As rich soil, which increased 4–10 times
when inoculated with metal resistant Bradyrhizobium sp. strain.
The present observations indicate that S. arlettae strain
NBRIEAG-6 effectively increased the bioavailability of As in the rhizosphere soils and also promoted the growth of B. juncea plants,
consequently increasing the total As uptakes of the plants and
decreasing the soil As content (Fig. 4c). Many reports have been
generated recently on the use of plants, assisted by metal tolerant
PGPRs for assisting metal phytoremediation [88–90]. This suggests
that this strain not only protects the plants against the inhibitory
effects of As but also effectively sequesters As in the root system.
Thus can be used to phytostabilize As in the plant root system.
4. Conclusions
We conclude that S. arlettae strain NBRIEAG-6 exhibited resistance to very high concentration of As and protects the plants
against the inhibitory effects of As through its plant growth promoting activity by producing IAA, ACC, siderophore and solubilizing
the phosphate. The presence of arsC genes confirmed the presence
of arsenate reductase activity which converts As(V) into As(III), that
may be sequestered in vacuoles. Thus over expression of this gene in
selected strain will further enhance the detoxification process. Furthermore, the isolated strain also enhanced the phytostabilization
of As in the root system. Therefore, the successful inoculation of this
bacterial strain may be especially important in hyperaccumulator
food crops like Brassica so that it could restrict the As accumulation in root system rather than the edible parts and the subsequent
entry in food chain.
Acknowledgements
Thanks are owed to The Director, CSIR-National Botanical
Research Institute, Lucknow for his kind support. SS thankfully
acknowledges CSIR for awarding SRF grant. This work was supported by Network Project (NWP-19) of Council of Scientific and
Industrial Research, Govt. of India.
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