bioacumulacion de mercurio y sintesis por enterobacterias pdf

8/18/2019 Bioacumulacion de Mercurio y Sintesis Por Enterobacterias PDF

1/4

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 mercurybioaccumulation with simultaneous synthesis of mercury nanoparticles. The culture conditions viz. pH

8.0 and lower concentration of mercury promotes synthesis of uniform sized 2–5 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 effluent and developing a green process for nanoparticles biosynthesis.

2010 Elsevier Ltd. All rights reserved.

1. Introduction

Nanoparticles are finding 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 causinga major environmental concern. Conventionally absorbents, ion ex-

change, reverse osmosis and electro-chemical treatment are used to

reducemercury level in industrial waste water (Chiarle et al., 2000).

However, these techniques are expensive and non-specific. Major

problem is caused due to unique property of mercury to enter into

vapor stage at room temperature (from Hg2+ to Hg0) (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-Döbler, 2005). Thus the ideal process for

mercury detoxification should be able to trap it as Hg2+ or as mer-

cury Hg0.

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 effluent. 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-

ism that 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 inoculum from 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: [email protected] (S.K. Khare).

Bioresource Technology 102 (2011) 4281–4284

Contents lists available at ScienceDirect

Bioresource Technology

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / b i o r t e c h

http://dx.doi.org/10.1016/j.biortech.2010.12.040mailto:[email protected]://dx.doi.org/10.1016/j.biortech.2010.12.040http://www.sciencedirect.com/science/journal/09608524http://www.elsevier.com/locate/biortechhttp://www.elsevier.com/locate/biortechhttp://www.sciencedirect.com/science/journal/09608524http://dx.doi.org/10.1016/j.biortech.2010.12.040mailto:[email protected]://dx.doi.org/10.1016/j.biortech.2010.12.040


2/4

Culture medium containing (g L 1): yeast extract, 3.0; peptone,

5.0; glucose, 5.0; NaCl, 2.5; MgSO47H2O, 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 A660 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 HgCl2 (final concentration) of filter sterile HgCl2 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 14,000 g 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 tetrahydroborateaccording 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 HgCl2 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 G2; 200 kV, USA). High resolution transmission

electron microscopy (HRTEM) and energy dispersive X-ray analysis

were done on the same bacterial thin film 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 HgCl2 was centrifuged at 14,000 g for 10 min at 4 C. The

pellet was washed thrice with Milli Q water and finally 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 chargingeffect.

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 fine 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 filtered through 0.45 l

Millipore filter. One drop of filtered 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 filtrate

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 profile of

the isolate in the medium containing 5 mg L 1 HgCl2. 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 (A660) in absence of HgCl2; [N],

bacterial growth (A660) in presence of 5 mg L 1 HgCl2; , residual mercury

concentration in culture media in presence of Enterobacter sp. cells; , residualmercury concentration in culture media in absence of Enterobacter sp. cells.

4282 A. Sinha, S.K. Khare / Bioresource Technology 102 (2011) 4281–4284


3/4

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 confirmed by their EDAX analysis (Supplementary data

Fig. S1a). EDAX signals confirmed that the accumulated particles

were indeed the mercury particles.

The accumulated mercury particles were further characterizedby 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 Hg2+ (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 reflections (002),

(221), (321), (141), (213) and (133) of monoclinic Hg3(PO4)2 (JCPDS

# 70–1798). 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) effluxing 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 andamount 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 HgCl2 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 sizeof 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 HgCl2 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-flash 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. TheTEM 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 Hg3(PO4)2, reconfirmed that the

remediated mercury is converted to mercuric phosphate nanopar-

ticles. Both the profiles 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 fromatoms to ecosystems. FEMS Micobiol. Rev. 27, 355–384.

Barkay, T., Wangner-Döbler, I., 2005. Microbial transformations of mercury:potentials, challenges, and achievements in controlling mercury toxicity in

the environment. Adv. Appl. Microbiol. 57, 1–52.Berry, C.C., De La Fuente, J.M., 2007. Special section on nanoparticles and QDs innanobiomedicine. IEEE Trans. Nanobiosci. 6, 261.

Brown, S., Sarikaya, M., Johnson, E., 2000. A genetic analysis of crystal growth. J. Mol.Biol. 299, 725–735.

Chiarle, S., Ratto, M., Rovatti, M., 2000. Mercury removal from water by ionexchange resins adsorption. Wat. Res. 34, 2971–2978.

Daniel, M.C., Astruc, D., 2004. Gold nanoparticles: assembly, supramolecularchemistry, quantum-size-related properties, and applications toward biology,catalysis, and nanotechnology. Chem. Rev. 104, 293–346.

David, G.F.X., Herbert, J., Wright, G.D.S., 1973. The ultrastructure of the pinealganglion in the ferret. J. Anat. 115, 79–97.

Devi, P.S.R., Kumar, S., Sudersan, M., 2006. Sorption of mercury on chemicallysynthesized polyaniline. J. Radioanal. Nucl. Chem. 269, 217–222.

Essa, A.M.M., Julian, D.J., Kidd, S.P., Brown, N.L., Hobman, J.L., 2003. Mercuryresistance determinants related to Tn 21, Tn1696 , and Tn5053 in Enterobacteriafrom the preantibiotic era. Antimicrob. Agents Chemother. 47, 1115–1119.

Gericke, M., Pinches, A., 2006. Biological synthesis of metal nanoparticles.Hydrometallurgy 83, 132–140.

Giersig, M., Henglein, A., 2000. Optical and chemical observations on gold-mercurynanoparticles in aqueous solution. J. Phys. Chem. B 104, 5056–5060.

A. Sinha, S.K. Khare / Bioresource Technology 102 (2011) 4281–4284 4283

http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://dx.doi.org/10.1016/j.biortech.2010.12.040http://dx.doi.org/10.1016/j.biortech.2010.12.040http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-http://-/?-


4/4

Gupta, A., Singh, R., Khare, S.K., Gupta, M.N., 2006. A solvent tolerant isolate of Enterobacter aerogenes. Bioresour. Technol. 97, 99–103.

Klaus-Joerger, T., Joerger, R., Olsson, E., Granqvist, C., 2001. G. Bacteria as workers inthe living factory: metal-accumulating bacteria and their potential for materialsscience. Trends Biotechnol. 19, 15–20.

Mandal, D., Bolander, M.E., Mukhopadhyay, D., Sarkar, G., Mukherjee, P., 2006. Theuse of microorganisms for the formation of metal nanoparticles and theirapplication. Appl. Microbiol. Biotechnol. 69, 485–492.

MHS 15 mercury hydride system, user’s guide, 2000. PerkinElmer Instruments,

Technical Documentation, PerkinElmer Bodenseewerk, Ueberlinger, pp. 3–9.

Nair, B., Pradeep, T., 2002. Coalescence of nanoclusters and formation of submicron crystallites assisted by Lactobacillus Strains. Cryst. Growth Des. 2,293–298.

Narayann, K.B., Sakthivel, N., 2010. Biological synthesis of metal nanoparticles bymicrobes. Adv. Colloid. Interface. Sci. 156, 1–13.

Nies, D.H., 1999. Microbial heavy-metal resistance. Appl. Microbiol. Biotechnol. 51,730–750.

Orton, B.R., Street, R.L.T., 1972. An X-ray diffraction study of liquid mercury between50 C and 150 C. J. Phys. C: Solid State Phys. 5, 2089–2097.

4284 A. Sinha, S.K. Khare / Bioresource Technology 102 (2011) 4281–4284

bioacumulacion de mercurio y sintesis por enterobacterias pdf

Documents