Short Communication

Mercury bioaccumulation and simultaneous nanoparticle synthesis by

Enterobacter sp. cells
Arvind Sinha, Sunil K. Khare

Enzyme and Microbial Biochemistry Lab, Department of Chemistry, Indian Institute of Technology, Delhi, Hauz Khas, New Delhi-110 016, India
a r t i c l e i n f o
Article history:
Received 17 October 2010
Received in revised form 7 December 2010
Accepted 8 December 2010
Available online 15 December 2010
Keywords:
Enterobacter sp.
Mercury bioremediation
Bioaccumulation
Mercury nanoparticle
a b s t r a c t
A mercury resistant strain of Enterobacter sp. is reported. The strain exhibited a novel property of mercury
bioaccumulation with simultaneous synthesis of mercury nanoparticles. The culture conditions viz. pH
8.0 and lower concentration of mercury promotes synthesis of uniform sized 25 nm, spherical and mon-
odispersed intracellular mercury nanoparticles. The remediated mercury trapped in the form of nanopar-
ticles is unable to vaporize back into the environment thus, overcoming the major drawback of mercury
remediation process. The mercury nanoparticles were recoverable. The nanoparticles have been charac-
terized by high resolution transmission electron microscopy, energy dispersive X-ray analysis, powder X-
ray diffraction and atomic force microscopy. The strain can be exploited for metal bioaccumulation from
environmental efuent and developing a green process for nanoparticles biosynthesis.
2010 Elsevier Ltd. All rights reserved.
1. Introduction
Nanoparticles are nding wide range of applications in biomed-
ical sciences, drug delivery, gene therapy, cell targeting, magnetics,
optics, mechanics, catalysis and energy science (Berry and De La
Fuente, 2007; Daniel and Astruc, 2004). Synthesis of nanoparticles
of different chemical compositions, sizes/shapes with controlled
monodispersity is one of the major challenges for their sustainable
use. Currently employed physical and chemical methods for the
synthesis of nanoparticles, have certain associated problems such
as stability, uncontrolled crystal growth and aggregation of the
nanoparticles (Klaus-Joerger et al., 2001). In this context, use of
microorganisms for the biosynthesis of nanoparticles has emerged
as a novel approach (Mandal et al., 2006; Narayann and Sakthivel,
2010).
Mercury is one of the third most toxic element (Nies, 1999).
Chlor-alkali, electronic industries and power plants discharge large
amount of mercury into the atmosphere and surface water causing
a major environmental concern. Conventionally absorbents, ion ex-
change, reverse osmosis and electro-chemical treatment are used to
reduce mercury level in industrial waste water (Chiarle et al., 2000).
However, these techniques are expensive and non-specic. Major
problem is caused due to unique property of mercury to enter into
vapor stage at room temperature (from Hg
2+
to Hg
0
) (Barkay et al.,
2003; Orton and Street, 1972). Thus, remediated mercury is often
recycled back into atmosphere in the form of mercury vapor.
Mercury remediating bacterial strains also have similar drawback
of volatilizing inorganic and organic mercury. Metallic mercury
produced by microbial reduction diffuses out of cells and vaporize
back to environment from the medium in case of Pseudomonas sp.
(Barkay and Wangner-Dbler, 2005). Thus the ideal process for
mercury detoxication should be able to trap it as Hg
2+
or as mer-
cury Hg
0
.
Present work explores mercury bioremediation with simulta-
neous synthesis of mercury nanoparticles by an Enterobacter sp.
strain (Gupta et al., 2006). The study demonstrates that metal
nanoparticles can be prepared from heavy metal containing media
or efuent. The process addresses two issues (i) bioremediation of
heavy metal pollutants (ii) nanobiosynthesis by a greener process.
2. Methods
2.1. Bacterial strain
Enterobacter sp. strain, an organic solvent-tolerant microorgan-
ismthat was isolated from soil was used in the present study (Gup-
ta et al., 2006). The culture was maintained at 4 C in agar slants
and sub-cultured at monthly intervals.
2.2. Inoculum and culture conditions
A loopful inoculumfrom the slant was introduced into the med-
ium containing (g L
1
): yeast extract 3.0; peptone, 5.0; NaCl, 2.5;
adjusted to pH 7.0 followed by incubation at 30 C and 120 rpm.
Twenty-four hour grown culture having OD 1.0 was used as seed
culture.
0960-8524/$ - see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2010.12.040

Corresponding author. Tel.: +91 11 26596533; fax: +91 11 26581102.

E-mail address: skhare@rocketmail.com (S.K. Khare).
Bioresource Technology 102 (2011) 42814284
Contents lists available at ScienceDirect
Bioresource Technology
j our nal homepage: www. el sevi er . com/ l ocat e/ bi or t ech
Culture medium containing (g L
1
): yeast extract, 3.0; peptone,
5.0; glucose, 5.0; NaCl, 2.5; MgSO
4
7H
2
O, 0.5; adjusted to pH 8.0
was inoculated with 1% seed culture. The inoculated medium
was incubated at 30 C with constant shaking at 120 rpm (Orbital
Rotary Shaker, Orbitech, India). The Enterobacter sp. growth was re-
corded at A
660
nm using double beam UV visible spectrophotome-
ter (Specord 200, Analyticjena, Germany).
2.3. Growth, residual mercury and biosynthesis of nanoparticles
5 mg L
1
HgCl
2
(nal concentration) of lter sterile HgCl
2
was
added into the culture medium prior to inoculation. Rest of the cul-
ture conditions were kept same as described in Section 2.2. The
sample was withdrawn periodically and processed for monitoring
(i) cell growth (ii) mercury concentration (iii) nanoparticle synthe-
sis. The cell growth was measured by recording the absorbance of
samples at 660 nm. Five mL of culture media was withdrawn
asceptically at regular time intervals, centrifuged at 14,000g for
10 min at 4 C. Supernatant was taken to estimate the residual
mercury using atomic absorption spectrophotometer (Per-
kinElmer MHS-15 Mercury/Hydride System, USA). The mercury
was estimated in each samples using sodium tetrahydroborate
according to the recommended conditions provided by the manu-
facturer (PerkinElmer MHS-15 Mercury/Hydride System, users
guide, 2000).
Effect of different parameters viz. pH, incubation time and me-
tal concentration on the growth, bioaccumulation and nanoparti-
cles synthesis by Enterobacter sp. was studied.
Cells were cultivated in culture media described previously, ex-
cept that one parameter was varied at a time. For pH, the culture
media was adjusted to pH 6.0, 7.0, 8.0 and 9.0, prior to inoculation.
For incubation time, the samples were ascetically withdrawn at
different time intervals 24, 48, 72, and 96 h (media pH was kept
8.0). The effect of mercury concentration was monitored by incor-
porating varying concentrations of HgCl
2
in the culture media
5 mg L
1
, 10 mg L
1
or 15 mg L
1
.
2.4. Characterization of mercury nanoparticle
2.4.1. Transmission electron microscopy (TEM)
Samples were processed for transmission electron microscopy
as per the procedure of David et al. (1973) to see the bioaccumula-
tion of mercury. Transmission electron micrographs were recorded
without regular double staining in TEM equipped with EDAX
(HRTEM, Technai G
2
; 200 kV, USA). High resolution transmission
electron microscopy (HRTEM) and energy dispersive X-ray analysis
were done on the same bacterial thin lm used for taking TEM
micrographs in nanoprobe mode.
2.4.2. X-ray photoelectron spectroscopy (XPS)
XPS was carried out to check the oxidation state of the accumu-
lated nanoparticles. Twenty mL of 96 h bacterial culture grown in 5
mg L
1
of HgCl
2
was centrifuged at 14,000g for 10 min at 4 C. The
pellet was washed thrice with Milli Q water and nally dissolved in
500 lL of Milli Q water. The resuspended cells were sonicated at a
frequency 24 KHz for 10 min. The sonicated culture was spreaded
uniformly over glass cover slip coated with 0.5% gelatin and dried
at room temperature. X-ray photoelectron spectroscopy (XPS)
was performed on Specs (SPECS GmbH, Berlin, Germany). The
photoelectrons were excited using an MgKa source of energy
1253.6 eV. The accuracy in binding energy determination
was 0.05 eV. The spectra obtained were calibrated to the binding
energy (BE) of C1s at 284.6 eV to compensate the surface charging
effect.
2.4.3. Powder X-ray diffraction (PXRD)
PXRD was done to identify the nature of mercury nanoparticles.
Cells were sonicated as described above and lysate was lyophilized
and crushed into ne powder and subjected to powder XRD (D2
Phaser, Bruker, Germany). Powder XRD pattern of cells grown in
absence of mercury was similarly recorded.
2.4.4. Recovery of mercury nanoparticle after sonication and high
resolution transmission electron microscopy (HRTEM)
The cells were sonicated and lysate was ltered through 0.45 l
Millipore lter. One drop of ltered lysate was loaded on carbon
coated grid, dried at room temperature and subjected to TEM/
HRTEM analysis for seeing the nature of the nanoparticles.
2.4.5. Atomic force microscopy (AFM)
The lysate was also subjected to AFM analysis for which ltrate
was spreaded uniformly on thin glass plate, dried at room temper-
ature. The AFM images were recorded on AFM system (Nanoscope
IIIa; Vecco Metrology Group, Santa Barbara, CA, USA) with a scan
rate of about 10.17 Hz to see the surface of the nanoparticles.
3. Results and discussion
3.1. Mercury bioaccumulation by Enterobacter sp.
We have previously reported a mercury resistant Enterobacter
sp. strain (Gupta et al., 2006). Fig. 1 shows the growth prole of
the isolate in the medium containing 5 mg L
1
HgCl
2
. Lag phase
was extended in presence of mercury, as compared to the control
(grown without mercury). Results show a continuous decrease in
mercury concentration simultaneous to the growth of Enterobacter
sp. Although the mercury resistance has been previously noted in
Enterobacteria (Essa et al., 2003), its use in remediation has never
been attempted.
0
1
2
3
4
5
0 24 48 72 96 120 144
Time (h)
C
o
n
c
e
n
t
r
a
t
i
o
n

o
f

m
e
r
c
u
r
y

(
m
g
L
-
1
)
0
1
2
3
4
5
A
6
6
0
Fig. 1. Growth, mercury bioremediation and transmission electron micrograph
(TEM) of Enterobacter sp. cells. Enterobacter sp. cells were grown in NB medium (pH
8.0) as described in Section 2.3. [], bacterial growth (A
660
) in absence of HgCl
2
; [N],
bacterial growth (A
660
) in presence of 5 mg L
1
HgCl
2
; , residual mercury
concentration in culture media in presence of Enterobacter sp. cells; , residual
mercury concentration in culture media in absence of Enterobacter sp. cells.
4282 A. Sinha, S.K. Khare / Bioresource Technology 102 (2011) 42814284
3.2. Characterization of accumulated mercury nanoparticles
The bioaccumulation of mercury in the cytoplasm was quite
evident in TEM micrographs of Enterobacter sp. cells, which were
further conrmed by their EDAX analysis (Supplementary data
Fig. S1a). EDAX signals conrmed that the accumulated particles
were indeed the mercury particles.
The accumulated mercury particles were further characterized
by HRTEM, XPS and XRD. The high resolution transmission electron
micrograph (HRTEM) image provides further insight into the struc-
ture of the intracellularly synthesized mercury nanoparticles (Sup-
plementary data Fig. S1b). The image exhibits lattice fringes with
d-spacing of 0.327 nm, which is consistent with the 0.327 nm sep-
aration between 031 planes in monoclinic mercuric phosphate.
The Hg 4f core level XPS is shown in Supplementary data Fig. S2.
The measured binding energy (101.05 eV, 4f
7/2
) of mercury in the
present work with the values available in the literature, indicate
the presence of mercury as Hg
2+
(Devi et al., 2006).
On comparing the powder XRD pattern of cells grown in pres-
ence and absence of mercury (Supplementary data Fig. S3) weak
diffraction peaks at d values 0.315, 0.301, 0.270, 0.256, 0.176,
0.179 can be recognized and assigned to the reections (002),
(221), (321), (141), (213) and (133) of monoclinic Hg
3
(PO
4
)
2
(JCPDS
# 701798). Thus all above characterization indicate that remedi-
ated mercury is accumulated nanosized mercuric phosphate
particles.
The mechanism of nanoparticles formation by microorganism is
yet to be fully understood. It is known that microbes detoxify the
metal by (i) efuxing it out (ii) accumulating in cytoplasm and
(iii) converting into less toxic form. The synthesis of nanosized par-
ticles around the metal center could be mediated through reduc-
tases, followed by aggregation with other cellular proteins (Nair
and Pradeep, 2002; Brown et al., 2000).
3.3. Effect of culture conditions on the nature of nanoparticles
The effect of various culture conditions viz. pH, growth time and
amount of mercury on the shape, size and numbers of nanoparti-
cles were investigated. Number of particles and their monodisper-
sibility increased with growth period. Very few small sized and
randomly dispersed particles were observed in 24 h grown cells.
Cells grown for 48, 72 and 96 h showed large number of spherical
nanoparticles which were uniformly dispersed in cytoplasm (data
not shown).
To see the effect of pH on synthesis of mercury nanoparticles,
Enterobacter cells were grown at 5 mg L
1
HgCl
2
in culture media
adjusted to different pH (Supplementary data Fig. S4). Particles of
irregular shape and size were formed at pH 6 (Supplementary
data Fig. S4a). Uniformly dispersed spherical nanoparticles were
seen on the cell wall as well as inside the cytoplasm at pH 7.0.
The particles were monodispersed, spherical in shape and size
of the particles ranged between 2 and 5 nm (Supplementary data
Fig. S4b). More intracellular nanoparticles were seen at pH 8.0.
(Supplementary data Fig. S4c). pH 9.0 led to extremely smaller
and less denser synthesis of nanoparticles (Supplementary data
Fig. S4d). The pH of the media is known to affect the size and dis-
tribution of nanoparticles. pH has been reported to critically affect
gold nanoparticles synthesis in Verticellum luteoalbum (Gericke
and Pinches, 2006).
The Enterobacter sp. was subjected to increasing amount of mer-
cury in the culture medium. The representative TEM micrographs
(Supplementary data Fig. S5) showed that nanoparticles synthesis
was concentration dependent and 5 mg L
1
HgCl
2
led to optimum
synthesis of nanoparticles. Concentration dependent gold nanopar-
ticle synthesis is previously reported in case of Verticellum luteoal-
bum (Gericke and Pinches, 2006).
3.4. Recovery of mercury nanoparticles by cell sonication
Mercury has intense plasmon absorption band. Such bands are
affected by a strong laser femto-ash with short relaxation time;
hence mercury nanoparticles are better suited for fast optical
devices (Giersig and Henglein, 2000). To assess the feasibility of
nanoparticles recovery, the Enterobacter cells containing intracellu-
lar mercury nanoparticles were subjected to ultrasonication. The
TEM micrograph of the cell lysate (Supplementary data Fig. S6a)
showed that the particles were recoverable and the average size
of recovered nanoparticles was 3.75 0.03 nm. These were
spherical in shape also evident from the AFM pictures. The mean
roughness as observed by AFM was found to be 1.575 nm (Supple-
mentary data Fig. S6b). Supplementary data Fig. S6c shows the
presence of clear lattice fringes with d-spacing of 0.355 and
0.32 nm, corresponding to 0.355 and 0.32 nm separation between
130 and 300 planes in monoclinic Hg
3
(PO
4
)
2
, reconrmed that the
remediated mercury is converted to mercuric phosphate nanopar-
ticles. Both the proles of recovered nanoparticles and those
present in intact cytoplasm were consistent and same.
4. Conclusions
The study thus proves that the Enterobacter sp. is a novel strain
which can be useful for mercury remediation and nanoparticle
synthesis. The remediated mercury cannot vaporize back to envi-
ronment and it is possible to recover it in nanoparticle form.
Acknowledgements
The research grant provided by Department of Biotechnology
(Govt. of India) for carrying out this study is gratefully acknowl-
edged. Author Arvind Sinha is grateful to University Grant Com-
mission, New Delhi for the award of Senior Research Fellowship.
Authors gratefully acknowledge the guidance and facilities for
nanoparticles provided by Prof. B.R. Mehta, Department of Physics,
IIT Delhi. The kind help given by Dr. Vidya Nand Singh in recording
and analyzing nanoparticles is also gratefully acknowledged.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.biortech.2010.12.040.
References
Barkay, T., Miller, S.M., Summers, A.O., 2003. Bacterial mercury resistance from
atoms to ecosystems. FEMS Micobiol. Rev. 27, 355384.
Barkay, T., Wangner-Dbler, I., 2005. Microbial transformations of mercury:
potentials, challenges, and achievements in controlling mercury toxicity in
the environment. Adv. Appl. Microbiol. 57, 152.
Berry, C.C., De La Fuente, J.M., 2007. Special section on nanoparticles and QDs in
nanobiomedicine. IEEE Trans. Nanobiosci. 6, 261.
Brown, S., Sarikaya, M., Johnson, E., 2000. A genetic analysis of crystal growth. J. Mol.
Biol. 299, 725735.
Chiarle, S., Ratto, M., Rovatti, M., 2000. Mercury removal from water by ion
exchange resins adsorption. Wat. Res. 34, 29712978.
Daniel, M.C., Astruc, D., 2004. Gold nanoparticles: assembly, supramolecular
chemistry, quantum-size-related properties, and applications toward biology,
catalysis, and nanotechnology. Chem. Rev. 104, 293346.
David, G.F.X., Herbert, J., Wright, G.D.S., 1973. The ultrastructure of the pineal
ganglion in the ferret. J. Anat. 115, 7997.
Devi, P.S.R., Kumar, S., Sudersan, M., 2006. Sorption of mercury on chemically
synthesized polyaniline. J. Radioanal. Nucl. Chem. 269, 217222.
Essa, A.M.M., Julian, D.J., Kidd, S.P., Brown, N.L., Hobman, J.L., 2003. Mercury
resistance determinants related to Tn21, Tn1696, and Tn5053 in Enterobacteria
from the preantibiotic era. Antimicrob. Agents Chemother. 47, 11151119.
Gericke, M., Pinches, A., 2006. Biological synthesis of metal nanoparticles.
Hydrometallurgy 83, 132140.
Giersig, M., Henglein, A., 2000. Optical and chemical observations on gold-mercury
nanoparticles in aqueous solution. J. Phys. Chem. B 104, 50565060.
A. Sinha, S.K. Khare / Bioresource Technology 102 (2011) 42814284 4283
Gupta, A., Singh, R., Khare, S.K., Gupta, M.N., 2006. A solvent tolerant isolate of
Enterobacter aerogenes. Bioresour. Technol. 97, 99103.
Klaus-Joerger, T., Joerger, R., Olsson, E., Granqvist, C., 2001. G. Bacteria as workers in
the living factory: metal-accumulating bacteria and their potential for materials
science. Trends Biotechnol. 19, 1520.
Mandal, D., Bolander, M.E., Mukhopadhyay, D., Sarkar, G., Mukherjee, P., 2006. The
use of microorganisms for the formation of metal nanoparticles and their
application. Appl. Microbiol. Biotechnol. 69, 485492.
MHS 15 mercury hydride system, users guide, 2000. PerkinElmer Instruments,
Technical Documentation, PerkinElmer Bodenseewerk, Ueberlinger, pp. 39.
Nair, B., Pradeep, T., 2002. Coalescence of nanoclusters and formation of
submicron crystallites assisted by Lactobacillus Strains. Cryst. Growth Des. 2,
293298.
Narayann, K.B., Sakthivel, N., 2010. Biological synthesis of metal nanoparticles by
microbes. Adv. Colloid. Interface. Sci. 156, 113.
Nies, D.H., 1999. Microbial heavy-metal resistance. Appl. Microbiol. Biotechnol. 51,
730750.
Orton, B.R., Street, R.L.T., 1972. An X-ray diffraction study of liquid mercury between
50 C and 150 C. J. Phys. C: Solid State Phys. 5, 20892097.
4284 A. Sinha, S.K. Khare / Bioresource Technology 102 (2011) 42814284