Chemosphere 95 (2014) 503510

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Chemosphere
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Removal of toxic Co-EDTA complex by a halophilic solar-salt-pan isolate

Pseudomonas aeruginosa SPB-1
A. Paraneeiswaran a, Sudhir K. Shukla b, T. Subba Rao b,, K. Prashanth a
a
Departartment of Biotechnology, Pondicherry University, Puducherry, India
b
Biofouling & Biolm Processes Section, Water & Steam Chemistry Division, BARC Facilities, Kalpakkam 603 102, India

h i g h l i g h t s


 P. aeruginosa SPB-1 was used in the bioremediation of [Co(III)-EDTA] .
-
 The strain removed the [Co(III)-EDTA] from the bulk liquid by adsorption.
 P. aeruginosa SPB-1 biomass could also be used as bio-resin.
 P. aeruginosa SPB-1 was found to be signicantly radio-resistant up to 300 Gy.

 This is the rst report on the biosorption of [Co(III)-EDTA] complex.

a r t i c l e i n f o a b s t r a c t

Article history: In this study, a promising bioremediation approach was developed to remove [Co(III)-EDTA] complex
Received 25 March 2013 that is generated during the waste management process. Though several studies have been reported
Received in revised form 12 September 2013 on bioremediation of cobalt, the removal of [Co(III)-EDTA] complex has not been tested. A
Accepted 26 September 2013
[Co(III)-EDTA] resistant bacterium, Pseudomonas aeruginosa SPB-1 was isolated from the solar-salt-
Available online 31 October 2013
pan and physical parameters were optimized for its growth. The various studies showed that the removal
of [Co(III)-EDTA] from the bulk liquid was due to the adsorption of the complex by the biomass. Using
Keywords:
absorption/desorption isotherm over a range of pH (18), the maximum adsorption of [Co(III)-EDTA]
Bioremediation
Cobalt
was found to be at pH 7.0 and maximum desorption from the biomass occurred at pH 1.0, thus rendering
[Co(III)-EDTA] an ion exchange property to P. aeruginosa SPB-1 biomass. P. aeruginosa SPB-1 biomass could be used as
Biosorption bio-resin that showed 80.4 3.27% adsorption capacity up to fourth cycle and the biomass was viable till
Pseudomonas aeruginosa the ninth cycle with 10.5 7.3% adsorption. Radiation tolerance potential i.e. D10 value for the strain was
found to be 300 Gy, which suggests the potential use of the bacterium in bioremediation of moderately
active nuclear waste.
2013 Elsevier Ltd. All rights reserved.

1. Introduction
Cobalt-60 is one of the most signicant radioisotopes among
the activated-corrosion products present in the waste generated
from some nuclear reactors (Lin, 2009). 60Co is the most prominent
radioisotope due to its longer half-life (5.26 years) and emission of
higher gamma energies (1.17 and 1.33 MeV) (Rufus et al., 2004).
Therefore, it is essential to remove cobalt to reduce the hazards
of radiation exposure to the maintenance personnel in the eld
conditions. Plenty of methods have been used at laboratory scale
to remove the cobalt from radioactive liquid waste, such as adsorp-
tion using activated carbon (Hete et al., 2012), montmorillonit
(Anjum and Datta, 2012), ion-exchange (Rengaraj and Moon,
Corresponding author. Tel.: +91 44 2748 0203; fax: +91 44 2748 0097.
E-mail address: subbarao@igcar.gov.in (T. Subba Rao).
2002; Yeon et al., 2003), nano-ltration (Szke et al., 2005), poly-
urethane foams (Rao et al., 1997), cobalt imprinted-polymer
(Bhaskarapillai et al., 2009) and Co(II)-selective modied Chitosan
(Nishad et al., 2012). Among the various methods reported, chem-
ical precipitation and adsorption methods are commonly used in
nuclear industries to remove cobalt (Rao et al., 1997). Though,
these methods have been used in nuclear industries they are not
efcient enough since they deliver low decontamination factor.
The decontamination factor is dened as the ratio of initial specic
activity to the nal specic activity of the waste solution. Addition-
ally ion-exchange resins are also used to remove cobalt from liquid
waste but their usage has been limited since their removal ef-
ciency in the presence of dissolved salts is low. One more method
has been reported that employs the mineral mixtures to separate
the cobalt but their efciency depends upon the presence of the

0045-6535/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.chemosphere.2013.09.107
504 A. Paraneeiswaran et al. / Chemosphere 95 (2014) 503510
cations, which reduce the efciency because the cations preoccupy
the site of cobalt (Rao et al., 1997).
During the decontamination operation, active Co(II) is re-
moved by loading onto cationic resin leading to the generation
of cobalt loaded active resin bed (Rufus et al., 2004). Chemical
precipitation involves the oxidation of Co(II) to Co(III) (Rao
et al., 1997). Many chelators are used to remove metal ions such
as oxalic acid, citric acid and ethylene diamine tetra acetic acid
(EDTA). These chemicals have shown a strong binding afnity
with cobalt and forms citrate, oxalate and EDTA complexes of co-
balt. Among which cobalt citrate and cobalt oxalate can easily be
removed by chemical precipitation but the removal of cobalt-
EDTA complex i.e. [Co(III)-EDTA] which is formed due to the oxi-
dation of [Co(II)-EDTA]2 is cumbersome. [Co(III)-EDTA]cannot
be removed by simple ltration and precipitation due to its high
stability and solubility (Szke et al., 2005). However, physico-
chemical methods are in use to remove the cobalt from nuclear
waste but consumption of high energy, high cost, generation of
large amounts of sludge and incomplete removal of metal ions
renders the physicochemical methods less procient (Gadd,
1990). To overcome these disadvantages, bioremediation ap-
proaches using heavy metal resistant microorganisms like bacte-
ria and fungus were carried out by earlier researchers (Yan and
Viraraghavan, 2003; Vijayaraghavan et al., 2005a; Pal et al.,
2006). In a specic report, it was inferred that the bacteria, fun-
gus and algae isolated from heavy metal polluted areas are the
ideal candidates for the bioremediation of heavy metals (Gadd,
2010). A number of studies have been carried out in order to uti-
lize microorganisms as metal bio-sorbents, however, reports on
microbial-biosorption of cobalt are scarce. Moreover, studies have
been carried out for the cobalt removal (Rashmi et al., 2004;
Vijayaraghavan et al., 2005a,b; Pal et al., 2006) but the resistance
of the microorganisms towards [Co(III)-EDTA] complex has not
been tested.
In the present study, a [Co(III)-EDTA] resistant bacteria strain
of Pseudomonas aeruginosa strain SPB-1 was isolated from solar salt
pond sediment samples at Marakkanam, Tamilnadu, India. The
growth conditions and the removal of [Co(III)-EDTA] from the
bulk aqueous solution were optimized. The study further evaluated
the bacteriums resistance to [Co(III)-EDTA] complex as well as its
potent application in bioremediation studies. We have also studied
the isolate radio-tolerance to claim its prospective use in the treat-
ment of moderate radioactive waste.
2. Materials and methods
2.1. Preparation of [Co(III)-EDTA]
[Co(III)-EDTA] was prepared based on the method of Taylor
and Jardine (1995) with few modications. For the preparation
of [Co(III)-EDTA] (15 mL), 2 g of Co(II)Cl26H2O, 3.2 g of Na2.
EDTA and 7.2 g of sodium acetate were added and heated to boil.
Then 3.2 mL of 7% H2O2 was added as an oxidizing agent with
continuous stirring. The solution was cooled to room tempera-
ture and 10 mL of 100% ethanol was added slowly with contin-
uous stirring. The precipitated crystals were washed thrice
with 100% ethanol and allowed to air dry. Crystals were then
dissolved in a small volume of double distilled water, till it
was completely solubilized. Then it was recrystallized in 100%
ethanol and subjected to two additional ethanol washes. The
crystals were then allowed to dry on low heat (40 C) until all
ethanol evaporated and stored at 4 C. The obtained product
was resuspended in double distilled water, ltered by using
0.22 lm lter and analyzed spectro-photometrically at 535 nm
for [Co(III)-EDTA].
2.2. Isolation of [Co(III)-EDTA] resistant bacteria from solar saltern
crystallizer pond
Soil samples (10 g) from the solar saltern crystallizer pond of
Marakkanam [Latitude (N) 12 200 : Longitude (E) 79 950 ) Southern
coast of the Bay of Bengal, India, were collected randomly from dif-
ferent locations of the salt pans and transferred to the laboratory in
sterilized poly bags under cold condition. 1 g of the soil sample was
suspended in 100 mL of sterile PBS and serially diluted. 100 lL of
the suspension was plated on acetate minimal agar comprised of
(1 L); 1 g of NH4Cl, 0.001 g of CaCl22H2O, 0.2 g of MgSO47H2O,
0.001 g of FeSO4, 5 g of CH3COONa and 0.5 g of K2HPO4, adjusted
to a nal pH 7.2 and was sterilized by autoclaving. Acetate minimal
agar amended with 1 mM [Co(III)-EDTA] was used for the isola-
tion of [Co(III)-EDTA] resistant bacteria. Fungal antibiotic cyclo-
sporine-A (10 lg mL1) was added to the media to avoid fungal
contamination. The plates were incubated at 30 C for 48 h. The
isolated pure cultures of bacteria were maintained as glycerol
stocks at 80 C until required.
2.3. Identication and molecular characterization of the bacterium
Bacterial genomic DNA was isolated based on the method as de-
scribed by (Sambrook and Russell, 2001). The isolated DNA was
then dissolved in 25 mL TE buffer (10 mM Tris, 1 mM EDTA; pH
8.0). 16S rRNA gene of bacteria was amplied using the universal
primers 16S-27F (50 AGAGTTTGATCMTGGCTCAG30 , M = A or C)
and 16S-1492R (50 ACGGCTACCTTGTTACGA 30 ). PCR was carried
out in a nal reaction volume of 25 lL in a thin wall PCR tube in
Eppendorf Thermal Cycler. The PCR reaction mixture was prepared
as follows: 7.5 lL of nuclease-free water, 12.5 lL 2  PCR master
mix (MBI Fermentas), 1.0 lL forward primer (10 pmol lL1),
1.0 lL reverse primer (10 pmol lL1), 3.0 lL diluted DNA
(30 ng lL1). The PCR conditions were as follows; initial denatur-
ation 95 C for 5 min, 30 cycles at 95 C for 1 min, 59 C for
1 min, 72 C for 2 min and nal extension of 72 C for 5 min. The
amplied PCR product was visualized under UV. Amplied PCR
products were excised and eluted from the gel and it was puried
using the QIA quick gel extraction kit (Qiagen). Puried amplicon
was then custom sequenced at Euron Genomics Private Limited,
Bengaluru facility. Partial 16S rRNA gene sequence information of
the amplicon obtained was then subjected to BLAST (Basic local
Alignment Search tool) analysis with non-redundant database of
National Council for Biotechnology Information (NCBI) GenBank.
The gene sequence was evaluated and sequence with highest sim-
ilarity was identied and deposited in the NCBI Genbank nucleo-
tide sequence database for accession number.
2.4. Analysis of in situ cobalt content of solar saltern soil
Soil samples were collected from the solar saltern crystallizer
pond and were analyzed to determine the cobalt content of the
soil. Samples were prepared according to the standard method as
described by Van Loon and Lichwa (1973). Briey, collected soil
sample was dried in hot air oven and sieved through muslin cloth.
The oven dried samples were kept at 400500 C in a mufed fur-
nace overnight to prepare ash. 1 g of ash was treated with Aqua-re-
gia (2 mL of 65% HNO3 + 6 mL of HCl). Hot plate was used to carry
out the digestion until dense fume evolved and clear solution was
obtained. The clear solution was ltered through lter paper
(Whatman No. 42). The ltrate was used for the determination of
cobalt by inductively-coupled plasma-atomic emission spectrome-
ter (ICP-AES, model: Ultima-2, Horiba JovinYvon, France).
 A. Paraneeiswaran et al. / Chemosphere 95 (2014) 503510
2.5. Survival of P. aeruginosa SPB-1 at low-dose radiation
Decontamination radioactive wastes are reported to have low-
level radiation, roughly 3.7  106 Bq L1, which is equal to
1.3 mGy (Rao et al., 1997). Therefore, survival potential of P. aeru-
ginosa SPB-1 in the presence of low-level radiation was evaluated
in the range of 10500 Gy. Radiation resistance was tested and
D10 value calculated based on the method described by Ito et al.
(1972). P. aeruginosa SPB-1 was grown in LB medium until it
reached the exponential growth. Mid log phase cells were har-
vested by centrifugation at 8000 rpm for 5 min. The pellets were
resuspended to a nal concentration of 1  108 cfu mL1 in
0.067 M phosphate buffered saline at pH 7.0. The suspension was
divided into 5 mL aliquots and exposed to 60Co source Gamma
Chamber 5000 [Board of Radiation and Isotope Technology (BRIT),
Mumbai, India], with dose rate of 2.8 Gy min1. After irradiation,
100 lL of each suspension was removed and diluted in the same
buffer and plated in LB agar plates. The number of CFU of SPB-1
was determined after 72 h of incubation.
2.6. Determination of minimum inhibitory concentration (MIC) of
[Co(III)-EDTA] against P. aeruginosa SPB1
Minimum tolerable concentration of a metal is dened as the
minimum quantity of a metal at which visible growth of the bacte-
ria is completely inhibited by the toxicity of the metal ion (Yilmaz,
2003). The MIC of [Co(III)-EDTA] against the P. aeruginosa SPB-1
was determined using the standard spread plate count method. A
wild type laboratory strain of P. aeruginosa was also included in
this experiment. Acetate minimal medium incorporated with
various concentrations of [Co (III)-EDTA] ranging from 0.5 mM
to 2.5 mM was used to determine MIC of [Co(III)-EDTA] against
P. aeruginosa SPB1. 100 lL of containing approximately 1  106 -
CFU mL1 bacteria were spread in triplicates on acetate minimal
media plates incorporated with different concentration of
[Co(III)-EDTA] and plates without [Co(III)-EDTA] was used as
a control. The plates were incubated at 37 C for 2472 h to ob-
serve the visible growth of the bacteria.
2.7. Growth kinetics of bacterium in the presence and absence of
[Co(III)-EDTA]
Bacterial growth was monitored in acetate minimal medium
with pH 6.0, pH 7.0 and pH 8.0 containing 1 mM [Co(III)-EDTA]
and without cobalt EDTA. 100 mL acetate minimal medium was
prepared, autoclaved and inoculated with overnight grown bacte-
rial culture (1:100) which had approximately 1  108 CFU mL1.
The culture was incubated at 37 C and 30 C in an orbital shaker
at 100 rpm and samples were collected at every 24 h time interval
up to 96 h. The growth of the bacterial culture was measured by
absorbance at 600 nm.
2.8. [Co (III)-EDTA] removal assay
Two parameters, temperature and pH were used to determine
the optimum conditions for the removal of [Co(III)-EDTA]. Two
temperature 30 C and 37 C were tested for optimum removal of
[Co(III)-EDTA] by P. aeruginosa SPB1. To determine the optimum
pH for the [Co(III)-EDTA] removal by P. aeruginosa SPB1, three
different pH concentrations were assayed such as 6.0, 7.0 and 8.0
using the acetate minimal medium. Removal was measured in
various combinations of two parameters to nd out optimum
growth condition to get efcient removal of [Co(III)-EDTA].
[Co(III) EDTA] removal capability from the bulk liquid was esti-
mated as follows. The bacterium was grown in acetate minimal
media incorporated with 1 mM [Co(III)-EDTA] in orbital rotary
505
shaker at 100 rpm. The inoculum having 1  106 cfu mL1 was
used to analyze the metal removal potential of bacterium. The
samples were collected at 24 h intervals up to 96 h and biomass
was removed by centrifugation at 12 000 rpm for 15 min. The
supernatant was ltered through 0.22 lm membrane lter and
the ltrate was analyzed spectro-photometrically at 535 nm for
the presence of remaining [Co(III)-EDTA] (Taylor and Jardine,
1995). In the above experiment, acetate minimal media amended
with 1 mM [Co(III)-EDTA] was used as a control and bacterium
inoculated in acetate minimal media was used as a test sample.
2.9. SEMEDAX analysis
Scanning electron microscopeEnergy dispersive X-ray analysis
(SEMEDAX) was done according to the protocol described else-
where with slight modications. [Co(III)-EDTA] loaded bacterial
biomass were xed with PBS (pH 7.2) containing 2% glutaralde-
hyde overnight at 4 C and they were washed in sterile distilled
water followed by dehydration in a graded alcohol series. Adsorbed
samples on cover slips were dried under an infrared lamp and were
attached to the aluminum stubs using carbon tape and sputter-
coated with carbon in a Balzers MED 010 unit (USA). Observations
were performed using a HITACHI S-3400 Scanning Electron Micro-
scope in high-vacuum mode at 10 kV and EDAX analysis was per-
formed using Thermo Noran System VI. Sample processing for
microscopy, SEM and EDAX analysis were performed at the central
instrumentation facility of Pondicherry University.
2.10. Adsorption/desorption assay for the removal of [Co(III)-EDTA]
To evaluate the adsorption potential of the bacterial biomass,
biosorption isotherm experiments were conducted at different
pH, different concentration of biomass, and contact time. Biosorp-
tion isotherm experiments were done using 25150 mg of biomass
with 1 mM [Co(III)-EDTA] at pH range 18 and contact time start-
ing from 1 to 12 h in an orbital shaker at 100 rpm. After 12 h of
contact time with [Co(III)-EDTA], biomass were harvested by cen-
trifuging at 12 000 rpm for 15 min. [Co(III)-EDTA] concentration
in the supernatant solution was analyzed spectrophotometrically
at 535 nm. To nd out the equilibrium attainment of biosorption,
a kinetic experiment was carried using the same method except
that the samples were collected at appropriate intervals. The
amount of [Co(III)-EDTA] absorbed by the bacterial biomass was
calculated by using the equation
Q e C o  C r  M  V
where Qe is the amount of [Co(III)-EDTA] uptake, Co and Cr are the
initial and nal concentration of [Co(III)-EDTA] in the supernatant
(mM), V is the volume of sample and M is the molecular mass of the
[Co(III)-EDTA].
In order to nd the recurrent reusability of biomass as an ion-
exchange resin to remove the [Co(III)-EDTA] complex, adsorption
experiments were conducted using 100 mg of biomass with 1 mM
[Co(III)-EDTA] at pH 7.0 followed by desorption at pH 1.0. Adsorp-
tion and desorption cycles were conducted continuously and the
percent residual adsorption capacity was calculated after each
cycle.
3. Results and discussion
3.1. Synthesis of [Co(III)-EDTA]
Absorption maxima for [Co(III)-EDTA] was checked by scan-
ning its absorbance in visible range i.e. 400600 nm, maximum
absorption for [Co(III)-EDTA] was observed at 535 nm. The
506 A. Paraneeiswaran et al. / Chemosphere 95 (2014) 503510

prepared samples showed an absorption maxima at 535 nm which
conrms the presence of [Co(III)-EDTA] in the sample (Supple-
mentary Fig. A).
3.2. Identication of bacterium and submission 16S rRNA gene
sequence
metal tolerance might be due to the unique strategies or resis-
tance/tolerance mechanisms exhibited by the bacterium.
Resistance to Co2+ in gram negative bacteria is based on the
trans-envelope efux system driven by a resistance nodulation cell
division (RND) transporter (Nies, 1999).

3.5. Effect of pH and temperature on growth kinetics of P. aeruginosa

[Co(III)-EDTA] resistant bacteria was isolated from solar
SPB1 in presence of [Co(III)-EDTA]
saltern soil that was able to grow in the presence of 1 mM
[Co(III)-EDTA]. This bacterium was identied by molecular
Fig. 1 shows the effect of [Co(III)-EDTA] on the growth of bac-
characterization as P. aeruginosa by analyzing 16S rRNA gene
teria at different pH and temperature conditions that was per-
sequence information. The 16S rRNA gene sequence information
formed in acetate minimal medium supplemented with 1 mM
of P. aeruginosa SPB-1 was deposited in NCBI Genbank nucleotide
[Co(III)-EDTA]. P. aeruginosa SPB1 manifested a different growth
sequence database with the accession number JQ809326 (Pseudo-
pattern in the presence and absence of [Co(III)-EDTA]. In the ab-
monas aeruginosa SPB-1).
sence of [Co(III)-EDTA] complex and at pH 7.0 and 8.0, the bacte-
rium exhibited a typical bacterial growth curve at both the
3.3. Cobalt content of solar saltern soil samples and halophilicity of P.
temperatures, 30 C as well as 37 C but there was no growth ob-
aeruginosa SPB1
served at pH 6.0 in either case. Since, the bacterium showed the
optimum growth at pH 8.0 and no growth at pH 6.0, it was inferred
Microorganisms exposed to a constant and continual environ-
that the P. aeruginosa SPB1 is an alkaliphilic bacterium isolate.
ment of various heavy metals at low concentration can undergo
On the other hand, the presence of 1 mM [Co (III)-EDTA] sub-
physiological and genetic adaptation (Gadd, 1993). Such changes
stantially inuenced the growth of P. aeruginosa SPB1 by extending
may bring selective advantage to the microbiota resulting in toler-
the lag phase of the growth up to 48 h. At both the temperatures
ance and adaptation of the microorganism to the higher concentra-
and pH 7.0 and 8.0, the stationary phase was attained at 72 h when
tions of the heavy metals and high salt concentration. To validate
compared to the their respective controls where stationary phase
this hypothesis, cobalt content of the solar saltern soil was ana-
was attained after 48 h. As observed in control study, the
lyzed. In situ cobalt concentration was found to be 0.32 mM and
bacterium showed optimum growth at pH 8.0 in the presence of
it is signicantly higher when compared to an earlier study
[Co(III)-EDTA] and relatively better growth at 37 C when
(Achyuthan et al., 2002). The authors reported that the concentra-
tion of Co2+ was in the range of 0.030.16 mM, however, they ana-
lyzed only the sediment samples collected from the tidal zones of
A 1.6 pH6 control
Marakkanam back waters. The constant cycle of evaporation and o
30 C pH6+CoEDTA
precipitation of seawater in the solar saltern area results in the 1.4 pH7 control
Bacterial growth (OD600nm)

pH7+CoEDTA
accumulation of heavy metals which may gradually increase over
1.2 pH8 control
a period of time. pH8+CoEDTA
The P. aeruginosa SPB1 strain was isolated from a solar-salt pan 1.0
and it was assumed that the strain is halophilic. A laboratory strain 0.8
of P. aeruginosa (WT) was also included in this study as a control.
Growth of P. aeruginosa SPB1 and P. aeruginosa WT were compared 0.6
at different salt concentration (Supplementary Fig. C). Three con- 0.4
centrations of NaCl were tested to observe its effect on its growth;
0.8 M (slight halophile), 3.5 M (moderate halophile) and 5 M (ex- 0.2
treme halophile) (Anton et al., 2000). The strain SPB1 showed the 0.0
substantial growth at 0.8 M (4.7%) and 3.5 M (20.5%) but no growth
at 5 M (29.2%) NaCl. Control also showed signicant growth in -0.2
0 24 48 72 96
both 0.8 M and 3.5 M, however, the growth was lesser than that Time (in Hours)
of P. aeruginosa SPB-1 strain. Therefore, P. aeruginosa SPB1 can be
considered as moderately halophilic bacterium. This study also
corroborates with the study of Anton et al. (2000), where bacteria
B 2.00 o
37 C
pH6 control
pH6 + CoEDTA
1.75
Bacterial growth (OD600nm)

isolates from a crystallizer ponds from solar salterns were found to pH7 control
pH7 + CoEDTA
show substantial growth up to 2025% NaCl concentration. 1.50
pH8 control
pH8 + CoEDTA

3.4. Minimum inhibitory concentration of [Co(III)-EDTA] for P. 1.25

aeruginosa SPB-1 1.00

MIC was used as an index to evaluate the tolerance prole of the 0.75
bacterium P. aeruginosa SPB-1 to [Co(III)-EDTA] complex. MIC of
0.50
[Co(III)-EDTA] for the isolate was found to be 2.5 mM (see Supple-
mentary Fig. B). On the other hand, MIC was found to be 1 mM for 0.25
the wild type strain of P. aeruginosa. This result clearly indicates
0.00
that P. aeruginosa SPB1 had 150% higher [Co(III)-EDTA] tolerance
0 24 48 72 96
than that of wild type P. aeruginosa. Thus, it had signicant
[Co(III)-EDTA] tolerance value as compared to the wild type labo- Time (Hours)
ratory strain of P. aeruginosa. The level of tolerance i.e., MIC of the Fig. 1. Growth curve of the P. aeruginosa SPB1 in the presence of 1 mM [Co(III)
bacterium was found to be higher than the value reported for E. coli EDTA] and at three different pH conditions viz., 6.0, 7.0 and 8.0 at (A) 30 C and (B)
for Co2+ in TRIS buffered medium (Nies, 1999). The variation in the 37 C. In control set no [Co(III)-EDTA] was added.
 A. Paraneeiswaran et al. / Chemosphere 95 (2014) 503510 507

compared to 30 C. At pH 7.0 and 8.0 extended lag phase indicated On the other hand, at the same pH, it showed slightly less [Co(III)-
that [Co(III)-EDTA] is highly toxic to cells therefore, P. aeruginosa EDTA] removal at 30 C as compared to 37 C, wherein 74% re-
SPB1 cells need longer time period to acclimatize physiologically to moval of [Co(III)-EDTA] was noticed. At pH 7.0, P. aeruginosa
[Co(III)-EDTA]. Moreover, growth of P. aeruginosa SPB1 at pH 8.0 SPB1 removed [Co(III)-EDTA] from the bulk medium at a slower
was found to be higher at all the time intervals tested, when com- rate in both the conditions i.e. at 30 C and 37 C. P. aeruginosa
pared to pH 7.0, both in the presence or absence of [Co (III)-EDTA] SPB1 removed 33.0 5.89% and 40 6.8% of [Co(III)-EDTA] at
complex. This observation was anticipated as seawater is used in 30 C and 37 C respectively. As shown in Fig. 2A and B there was
the Marakkanam solar-salt-pan and pH of seawater is known to no signicant removal of [Co(III)-EDTA] by the bacterium when
be P8.0. At pH 6.0, the bacterium did not show any substantial the assay was done at pH 6.0. This observation at pH 6.0 was antic-
growth in any of the test conditions. This indicates that pH 6 6.0 ipated since P. aeruginosa SPB1 growth studies in the presence of
had a strong inhibitory effect on P. aeruginosa SPB1 and cannot [Co(III)-EDTA], showed no growth, even in control experimental
be used for experimental conditions. sets.

3.6. Effect of pH and temperature on [Co(III)-EDTA] removal by P.

3.7. Effect of amount of biomass and contact time on adsorption of
aeruginosa SPB1
[Co(III)-EDTA]

Effect of different pH conditions and two incubation tempera-

Microorganisms commonly use three different mechanisms to
tures on the [Co(III)-EDTA] complex removal by P. aeruginosa
remove heavy metals viz., biosorption, bioaccumulation and
SPB1 were investigated. Fig. 2 shows that P. aeruginosa SPB1 re-
anaerobic reduction (Ahalya et al., 2003). SEMEDAX analysis of
moved the [Co(III)-EDTA] complex from the acetate minimal
P. aeruginosa SPB1 cell biomass grown in the presence of 1 mM
medium at a higher rate when the assay was done at pH 8.0 as
[Co(III)-EDTA] shows that there was a signicant increase in the
compared to pH 7.0 and pH 6.0 over the period of 96 h at 30 C
presence of cobalt in biomass which signies that [Co(III)-EDTA]
and 37 C. At pH 8.0 and 37 C, the concentration of [Co(III)-EDTA]
gets absorbed on biomass (Supplementary Table 1). To check the
was reduced from an initial concentration of 1 mM to 180 lM
effect of bacterial biomass concentration on the adsorption of
within 72 h, i.e. P. aeruginosa SPB1 removed 82% [Co(III)-EDTA].
[Co(III)-EDTA], an experiment was carried out using 25150 mg
of dry biomass in phosphate buffer saline (PBS). Fig. 3A illustrates
that the absorption of [Co(III)-EDTA] showed a linear increased
A 1.2
o
30 C
pH6
pH7
pH6 control
pH7 control
pH6 + SPB1
pH7 + SPB1 1.2
pH8 pH8 control pH8 + SPB1 A 3.5 SPB1 WT
Conc. of [Co(III)EDTA] (mM)
Planktonic growth (OD600)

Adsorbed [Co(III)EDTA] - (mg/g)

1.0 1.0
3.0
0.8 0.8
2.5
0.6 0.6
2.0

0.4 0.4 1.5

0.2 0.2 1.0

0.0 0.0 0.5

0 24 48 72 96 0.0
Time (hours)
0 25 50 75 100 125 150
Amount of biomass (mg)
pH6 pH6 control pH6 + PA SPB1
B 1.2
o
37 C pH7 pH7 control pH7 + PA SPB1 1.2
pH8 pH8 control pH8 + PA SPB1
B
3.0
Planktonic growth (OD600)

Conc. of [Co(III)EDTA] (mM)

1.0
Adsobed [Co(III)EDTA] - (mg/g)

1.0

2.5
0.8 0.8

2.0
0.6 0.6

1.5
0.4 0.4

1.0
0.2 0.2

0.5
0.0 0.0

0 24 48 72 96 0.0
Time (hours) 0 1 2 3 4 5 6 7 8 9 10 11 12 13

Fig. 2. Removal of [Co(III)-EDTA] by P. aeruginosa SPB1 at (A) 30 C. (B) 37 C. Solid Time (Hours)
lines represent the planktonic growth of P. aeruginosa SPB1. Dotted lines with
empty legends show blank i.e. growth medium containing 1 mM [Co(III) EDTA] at Fig. 3. (A) Effect of amount of biomass on adsorption of [Co(III)-EDTA] under
pH 6.0, 7.0 and 8.0. Figure legends with a plus (+) symbol represents tests at pH 6.0, neutral pH condition and 37 C. (B) Effect of contact time on the adsorption of
7.0 and 8.0 respectively. [Co(III)-EDTA] on xed amount of biomass.
508 A. Paraneeiswaran et al. / Chemosphere 95 (2014) 503510

with an increase in bacterial biomass up to 100 mg. Further in- 100 100
crease in biomass content did not show any signicant enhance- 90
Adsorption of [Co(III)-EDTA]

% Desorption of [Co(III)-EDTA]
90

% Adsorption of [Co(III)-EDTA]
Desorption of [Co(III)-EDTA]
ment in the adsorption of [Co(III)-EDTA] complex. On the other
80 80
hand wild type P. aeruginosa did not show signicant adsorption
at any of amount biomass when compared to SPB1 isolate. In case 70 70
of P. aeruginosa SPB1, 100 mg of biomass showed the absorption of 60 60
2.9 0.37 mg g1 as compared to maximum absorption of
50 50
0.70 0.04 mg g1 in P. aeruginosa WT (Fig. 3A).
Cell wall structure of a bacterial cell exhibits both anionic and 40 40
cationic functional moieties such as amino, phosphate, hydroxyl 30 30
and carboxyl groups which may act as active sites for [Co(III)- 20 20
EDTA] binding (Beveridge and Murray, 1980). Initially, increase
10 10
in biomass also increased the number of active sites present on
the biomass thereby resulting in a linear enhancement in 0 0
metalcomplex adsorption. When the amount of biomass was 0 1 2 3 4 5 6 7 8 9
increased >100 mg mL1, it caused the agglomeration of biomass pH
and homogenous suspension could not be obtained. An agglomer-
Fig. 4. Effect of different pH conditions on the adsorption and desorption of [Co(III)-
ation of biomass could result in a decrease in the number of active
EDTA] using a xed amount of biomass i.e. 100 mg mL1.
sites present resulting in a saturation stage. Thus, it could be in-
ferred that increase in biomass did not show any effect since the
the solution will enhance the ionization of Co-EDTA complex. At a
active sites are masked and not available for binding.
lower pH, ionization of the complex [Co(III)-EDTA] is reduced,
The effect of contact time on the adsorption of [Co(III)-EDTA]
thereby resulting in less sorption capacity of biomass towards
was monitored at various time intervals to nd out the optimum
[Co(III)-EDTA]. On the contrary, at higher pH, competition by ex-
time needed for [Co(III)-EDTA] removal from bulk liquid for a gi-
cess OH- takes place that binds the adsorbent and reducing the
ven amount of biomass. Fig. 3B shows that initially there was a
[Co(III)-EDTA] adsorption.
sharp increase in the adsorption of [Co(III)-EDTA] over a xed
amount of biomass i.e. 100 mg mL1. This amount of biomass
was chosen as it was found to be optimum for the adsorption
3.9. Effect of recurrent use of biomass as resin on adsorption capacity
experiments (see Fig. 3A). After 5 h, the rate of the adsorption grad-
ually declined and approached towards a saturation stage. The
Continuous adsorption and desorption experiments were car-
equilibrium stage was attained after 5 h, and there was no signi-
ried out in a batch system to monitor the possibility of repeated
cant increase in adsorption thereafter, even when monitored up to
use of P. aeruginosa SPB1 biomass as an ionexchange resin for
12 h. A possible explanation for this observation is as follows; as
the removal of [Co(III)-EDTA]. Fig. 5 shows that biomass retains
the contact time was increased there was an accumulation of
the stability up to fourth cycle as the bio-resin, showed
[Co(III)-EDTA] on the active sites of biomass. Once all the active
80.4 3.27% of residual adsorption capacity of [Co(III)-EDTA]
sites were occupied by [Co(III)-EDTA] and saturation stage was
complex as compared to the biomass used in the rst cycle. After
accomplished, there was no further binding of [Co(III)-EDTA].
fourth cycle residual adsorption capacity was reduced gradually,
at the end of the seventh cycle it reached 42 5.8%. After the sev-
3.8. Effect of pH on adsorption and desorption of [Co(III)-EDTA] to P.
enth cycle, there was a signicant decrease in the adsorption and
aeruginosa SPB1 biomass
only 10.5 7.3% of residual adsorption capacity retained at the
end of ninth cycle. The lower pH solution (pH 1.0) might modify
pH of the solution is a critical factor in the determination of the
the metal binding sites of bio-resin, therefore a signicant reduc-
adsorption/desorption capacity of any compound to bacterial bio-
tion in the complex-binding capacity was observed.
mass (Vijayaraghavan and Yun, 2008). Therefore, the inuence of
pH on the adsorption/desorption capacity of [Co(III)-EDTA] com-
plex to P. aeruginosa SPB1 was studied. To study the effect of pH
on adsorption mechanism, experiments were carried out with sus- 100
pensions having a different pH with the concentration of 1 mM
[Co(III)-EDTA]. Fig. 4 shows changes in adsorption/desorption
% Residual adsorption

80
with change in pH. Thus there was no signicant adsorption of
capacity of biomass

[Co(III)-EDTA] in the range of pH 1.04.0. As the pH increased

to more than 4, removal of [Co(III)-EDTA] from the bulk liquid 60
drastically increased up to pH 7.0. The removal of [Co(III)-EDTA]
complex was approximately 65.5 3.95% and 78.4 12.6% at the
pH 6.0 and 7.0, respectively. However, this enhancement in the 40
adsorption of [Co(III)-EDTA] was observed in the range of pH
47 and a signicant decline was observed in absorption of
[Co(III)-EDTA] at pH 8.0. On the contrary, desorption of [Co(III)- 20
EDTA] from the metal-complex loaded biomass was found to be
maximum at pH 1.0. Desorption efciency of biomass at pH 1.0
0
was 93.2 4.2%. From the above data, it can be inferred that the
0 1 2 3 4 5 6 7 8 9 10
maximum adsorption of [Co(III)-EDTA] took place in neutral con-
dition and the maximum desorption of [Co(III)-EDTA] from the No. of cycles (N)
metal-complex loaded biomass in strong acidic conditions. Fig. 5. Effect of recurrent use of biomass as resin on percent residual adsorption
The binding of the [Co(III)-EDTA] to bacterial biomass at vari- capacity of biomass. Percentage residual adsorption capacity was monitored up to
ous pH conditions can be explained as follows; increasing the pH of 9th cycle.
 A. Paraneeiswaran et al. / Chemosphere 95 (2014) 503510
8 Acknowledgments
10
Viable counts per ml
7
10
6
10
5 Supplementary data associated with this article can be found, in
10
4
10
0 100 200 300 400
Dose (in Gy)
Fig. 6. Survival curve of P. aeruginosa SPB1 exposed to different doses of gamma
radiation (dose rate of 2.8 Gy min1).
3.10. Evaluation of radio-resistance of P. aeruginosa SPB-1
The radio-resistance of many bacteria found to be closely
associated with the functions that are necessary to survive under
prolonged desiccation (Makarova et al., 2001). Since P. aeruginosa
SPB1 was isolated from a location where a frequent hydration
and desiccation cycle takes place, we investigated whether the
isolate has any enhanced radio-resistance/tolerance. Fig. 6 shows
the survival curve of P. aeruginosa SPB-1 to gamma radiation. D10
value for P. aeruginosa SPB1 found to be 300 Gy which indi-
cates that this bacterial isolate is radio-tolerant enough to sur-
vive in a moderate radiation condition of nuclear waste (Rao
et al., 1997).
Generally bacteria inhabiting the high saline conditions do cope
with extreme changes in salinity, temperature, desiccation and
natural radiation (Demergasso et al., 2008). Since this particular
geographic region of the solar saltern pond has been undergoing
constant cycles of evaporation and hydration over a very long per-
iod of time, it could result in the accumulation of various heavy
metals. Therefore, microorganisms isolated from saltern pond can
be anticipated to be tolerant towards heavy metals (Anyim et al.,
2012). This tolerance property can be utilized to examine the
removal of heavy metals. In such stress conditions, many genotypic
and phenotypic changes are also likely to occur at the species level
(Parsons, 1991). Therefore, we suggest detailed studies at the
molecular level to explore the underlying mechanism of tolerance
property acquired by P. aeruginosa SPB-1 isolated from the unique
sampling site. To the best of our knowledge, this work is the rst
report on the removal of [Co(III)-EDTA] complex by any
microorganism.
4. Conclusion
This study shows the potential utilization of a novel P. aerugin-
osa SPB-1 isolate in the bioremediation of [Co(III)-EDTA]. Studies
showed that the bacterial strain removes the [Co(III)-EDTA]
complex from the bulk liquid by adsorption phenomenon. We
optimized conditions for the removal of the [Co(III)-EDTA] com-
plex using P. aeruginosa SPB-1 biomass which acted as a bio-resin.
Since P. aeruginosa SPB-1 was found to survive in moderate level
radiation, we suggest that this bacterium could be an ideal candi-
date for the prospective use in bioremediation studies in nuclear
industry.
509
Authors thank Dr. R. Puspalatha, WSCD, for providing radiation
treatment facilities. Authors also acknowledge Dr. V.P. Venugopa-
lan, Mr. V.S. Sathyaseelan and Dr. B. Anupkumar, WSCD, for their
helpful discussions and suggestions during the preparation of the
manuscript.
Appendix A. Supplementary material
the online version, at http://dx.doi.org/10.1016/j.chemosphere.
2013.09.107.
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