Bioresource Technology 102 (2011) 4281–4284

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Bioresource Technology

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Short Communication

Mercury bioaccumulation and simultaneous nanoparticle synthesis byEnterobacter 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 a b s t r a c t

Article history:Received 17 October 2010Received in revised form 7 December 2010Accepted 8 December 2010Available online 15 December 2010

Keywords:Enterobacter sp.Mercury bioremediationBioaccumulationMercury nanoparticle

0960-8524/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.biortech.2010.12.040

⇑ Corresponding author. Tel.: +91 11 26596533; faxE-mail address: skhare@rocketmail.com (S.K. Khar

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. pH8.0 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 mercuryremediation 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 fromenvironmental 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 LaFuente, 2007; Daniel and Astruc, 2004). Synthesis of nanoparticlesof different chemical compositions, sizes/shapes with controlledmonodispersity is one of the major challenges for their sustainableuse. Currently employed physical and chemical methods for thesynthesis of nanoparticles, have certain associated problems suchas stability, uncontrolled crystal growth and aggregation of thenanoparticles (Klaus-Joerger et al., 2001). In this context, use ofmicroorganisms for the biosynthesis of nanoparticles has emergedas 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 largeamount 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 toreduce mercury level in industrial waste water (Chiarle et al., 2000).However, these techniques are expensive and non-specific. Majorproblem is caused due to unique property of mercury to enter intovapor stage at room temperature (from Hg2+ to Hg0) (Barkay et al.,2003; Orton and Street, 1972). Thus, remediated mercury is oftenrecycled back into atmosphere in the form of mercury vapor.Mercury remediating bacterial strains also have similar drawback

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of volatilizing inorganic and organic mercury. Metallic mercuryproduced by microbial reduction diffuses out of cells and vaporizeback to environment from the medium in case of Pseudomonas sp.(Barkay and Wangner-Döbler, 2005). Thus the ideal process formercury 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 metalnanoparticles can be prepared from heavy metal containing mediaor effluent. The process addresses two issues (i) bioremediation ofheavy 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 slantsand 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 seedculture.

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Culture medium containing (g L�1): yeast extract, 3.0; peptone,5.0; glucose, 5.0; NaCl, 2.5; MgSO4�7H2O, 0.5; adjusted to pH 8.0was inoculated with 1% seed culture. The inoculated mediumwas incubated at 30 �C with constant shaking at 120 rpm (OrbitalRotary 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 wasadded into the culture medium prior to inoculation. Rest of the cul-ture conditions were kept same as described in Section 2.2. Thesample 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 ofsamples at 660 nm. Five mL of culture media was withdrawnasceptically at regular time intervals, centrifuged at 14,000g for10 min at 4 �C. Supernatant was taken to estimate the residualmercury using atomic absorption spectrophotometer (Per-kin�Elmer MHS-15 Mercury/Hydride System, USA). The mercurywas estimated in each samples using sodium tetrahydroborateaccording to the recommended conditions provided by the manu-facturer (Perkin�Elmer MHS-15 Mercury/Hydride System, usersguide, 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 culturemedia was adjusted to pH 6.0, 7.0, 8.0 and 9.0, prior to inoculation.For incubation time, the samples were ascetically withdrawn atdifferent time intervals 24, 48, 72, and 96 h (media pH was kept8.0). The effect of mercury concentration was monitored by incor-porating varying concentrations of HgCl2 in the culture media5 mg L�1, 10 mg L�1 or 15 mg L�1.

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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 recordedwithout regular double staining in TEM equipped with EDAX(HRTEM, Technai G2; 200 kV, USA). High resolution transmissionelectron microscopy (HRTEM) and energy dispersive X-ray analysiswere done on the same bacterial thin film used for taking TEMmicrographs in nanoprobe mode.

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Fig. 1. Growth, mercury bioremediation and transmission electron micrograph(TEM) of Enterobacter sp. cells. Enterobacter sp. cells were grown in NB medium (pH8.0) 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 mercuryconcentration in culture media in presence of Enterobacter sp. cells; , residualmercury concentration in culture media in absence of Enterobacter sp. cells.

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 5mg L�1 of HgCl2 was centrifuged at 14,000g for 10 min at 4 �C. Thepellet was washed thrice with Milli Q water and finally dissolved in500 lL of Milli Q water. The resuspended cells were sonicated at afrequency 24 KHz for 10 min. The sonicated culture was spreadeduniformly over glass cover slip coated with 0.5% gelatin and driedat room temperature. X-ray photoelectron spectroscopy (XPS)was performed on Specs (SPECS GmbH, Berlin, Germany). Thephotoelectrons were excited using an MgKa source of energy1253.6 eV. The accuracy in binding energy determinationwas 0.05 eV. The spectra obtained were calibrated to the bindingenergy (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 lyophilizedand crushed into fine powder and subjected to powder XRD (D2Phaser, Bruker, Germany). Powder XRD pattern of cells grown inabsence of mercury was similarly recorded.

2.4.4. Recovery of mercury nanoparticle after sonication and highresolution transmission electron microscopy (HRTEM)

The cells were sonicated and lysate was filtered through 0.45 lMillipore filter. One drop of filtered lysate was loaded on carboncoated 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 (NanoscopeIIIa; Vecco Metrology Group, Santa Barbara, CA, USA) with a scanrate 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 Enterobactersp. strain (Gupta et al., 2006). Fig. 1 shows the growth profile ofthe isolate in the medium containing 5 mg L�1 HgCl2. Lag phasewas extended in presence of mercury, as compared to the control(grown without mercury). Results show a continuous decrease inmercury concentration simultaneous to the growth of Enterobactersp. Although the mercury resistance has been previously noted inEnterobacteria (Essa et al., 2003), its use in remediation has neverbeen attempted.

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3.2. Characterization of accumulated mercury nanoparticles

The bioaccumulation of mercury in the cytoplasm was quiteevident in TEM micrographs of Enterobacter sp. cells, which werefurther confirmed by their EDAX analysis (Supplementary dataFig. S1a). EDAX signals confirmed that the accumulated particleswere indeed the mercury particles.

The accumulated mercury particles were further characterizedby HRTEM, XPS and XRD. The high resolution transmission electronmicrograph (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 withd-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, 4f7/2) of mercury in thepresent work with the values available in the literature, indicatethe 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) weakdiffraction 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 phosphateparticles.

The mechanism of nanoparticles formation by microorganism isyet to be fully understood. It is known that microbes detoxify themetal 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 (Nairand 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 andrandomly dispersed particles were observed in 24 h grown cells.Cells grown for 48, 72 and 96 h showed large number of sphericalnanoparticles which were uniformly dispersed in cytoplasm (datanot shown).

To see the effect of pH on synthesis of mercury nanoparticles,Enterobacter cells were grown at 5 mg L�1 HgCl2 in culture mediaadjusted to different pH (Supplementary data Fig. S4). Particles ofirregular shape and size were formed at pH 6 (Supplementarydata Fig. S4a). Uniformly dispersed spherical nanoparticles wereseen 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 dataFig. S4b). More intracellular nanoparticles were seen at pH 8.0.(Supplementary data Fig. S4c). pH 9.0 led to extremely smallerand less denser synthesis of nanoparticles (Supplementary dataFig. S4d). The pH of the media is known to affect the size and dis-tribution of nanoparticles. pH has been reported to critically affectgold nanoparticles synthesis in Verticellum luteoalbum (Gerickeand 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 synthesiswas concentration dependent and 5 mg L�1 HgCl2 led to optimumsynthesis 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 areaffected by a strong laser femto-flash with short relaxation time;hence mercury nanoparticles are better suited for fast opticaldevices (Giersig and Henglein, 2000). To assess the feasibility ofnanoparticles 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 sizeof recovered nanoparticles was 3.75 ± 0.03 nm. These werespherical in shape also evident from the AFM pictures. The meanroughness as observed by AFM was found to be 1.575 nm (Supple-mentary data Fig. S6b). Supplementary data Fig. S6c shows thepresence of clear lattice fringes with d-spacing of 0.355 and0.32 nm, corresponding to 0.355 and 0.32 nm separation between130 and 300 planes in monoclinic Hg3(PO4)2, reconfirmed that theremediated mercury is converted to mercuric phosphate nanopar-ticles. Both the profiles of recovered nanoparticles and thosepresent in intact cytoplasm were consistent and same.

4. Conclusions

The study thus proves that the Enterobacter sp. is a novel strainwhich can be useful for mercury remediation and nanoparticlesynthesis. 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 fornanoparticles provided by Prof. B.R. Mehta, Department of Physics,IIT Delhi. The kind help given by Dr. Vidya Nand Singh in recordingand analyzing nanoparticles is also gratefully acknowledged.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.biortech.2010.12.040.

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