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Mercury bioaccumulation and simultaneous nanoparticle synthesis by Enterobacter sp. cells
Bioresource Technology 102 (2011) 4281–4284
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
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 2–5 nm, spherical and monodispersed intracellular mercury nanoparticles. The remediated mercury trapped in the form of nanoparticles 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 characterized by high resolution transmission electron microscopy, energy dispersive X-ray analysis, powder Xray diffraction and atomic force microscopy. The strain can be exploited for metal bioaccumulation from environmental effluent and developing a green process for nanoparticles biosynthesis. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Nanoparticles are finding wide range of applications in biomedical 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 exchange, 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-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 ⇑ Corresponding author. Tel.: +91 11 26596533; fax: +91 11 26581102. E-mail address: [email protected] (S.K. Khare). 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.12.040
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 mercury Hg0. Present work explores mercury bioremediation with simultaneous 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 microorganism that was isolated from soil was used in the present study (Gupta 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 medium 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.
A. Sinha, S.K. Khare / Bioresource Technology 102 (2011) 4281–4284
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 recorded at A660 nm using double beam UV visible spectrophotometer (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 culture 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 synthesis. 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 (Perkin Elmer MHS-15 Mercury/Hydride System, USA). The mercury was estimated in each samples using sodium tetrahydroborate according to the recommended conditions provided by the manufacturer (Perkin Elmer MHS-15 Mercury/Hydride System, users guide, 2000). Effect of different parameters viz. pH, incubation time and metal concentration on the growth, bioaccumulation and nanoparticles synthesis by Enterobacter sp. was studied. Cells were cultivated in culture media described previously, except 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 incorporating varying concentrations of HgCl2 in the culture media 5 mg L 1, 10 mg L 1 or 15 mg L 1.
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 temperature. 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.
2.4.2. X-ray photoelectron spectroscopy (XPS) XPS was carried out to check the oxidation state of the accumulated nanoparticles. Twenty mL of 96 h bacterial culture grown in 5 mg L 1 of HgCl2 was centrifuged at 14,000g 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 charging effect.
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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 bioaccumulation 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.
Concentration of mercury (mgL )
2.4. Characterization of mercury nanoparticle
A660
4282
0
0
0
24
48
72 Time (h)
96
120
144
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; , residual mercury concentration in culture media in absence of Enterobacter sp. cells.
A. Sinha, S.K. Khare / Bioresource Technology 102 (2011) 4281–4284
4283
3.2. Characterization of accumulated mercury nanoparticles
3.4. Recovery of mercury nanoparticles by cell sonication
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 characterized by HRTEM, XPS and XRD. The high resolution transmission electron micrograph (HRTEM) image provides further insight into the structure of the intracellularly synthesized mercury nanoparticles (Supplementary data Fig. S1b). The image exhibits lattice fringes with d-spacing of 0.327 nm, which is consistent with the 0.327 nm separation 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 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 presence 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 remediated 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 particles around the metal center could be mediated through reductases, followed by aggregation with other cellular proteins (Nair and Pradeep, 2002; Brown et al., 2000).
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 intracellular 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 (Supplementary 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 nanoparticles. Both the profiles of recovered nanoparticles and those present in intact cytoplasm were consistent and same.
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 nanoparticles were investigated. Number of particles and their monodispersibility 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 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 distribution 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 mercury 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 nanoparticle synthesis is previously reported in case of Verticellum luteoalbum (Gericke and Pinches, 2006).
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 environment 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 acknowledged. Author Arvind Sinha is grateful to University Grant Commission, 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, 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 in nanobiomedicine. 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 ion exchange resins adsorption. Wat. Res. 34, 2971–2978. 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, 293–346. David, G.F.X., Herbert, J., Wright, G.D.S., 1973. The ultrastructure of the pineal ganglion in the ferret. J. Anat. 115, 79–97. Devi, P.S.R., Kumar, S., Sudersan, M., 2006. Sorption of mercury on chemically synthesized 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. Mercury resistance determinants related to Tn21, Tn1696, and Tn5053 in Enterobacteria from 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-mercury nanoparticles in aqueous solution. J. Phys. Chem. B 104, 5056–5060.
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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 in the living factory: metal-accumulating bacteria and their potential for materials science. Trends Biotechnol. 19, 15–20. 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, 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 by microbes. 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 between 50 C and 150 C. J. Phys. C: Solid State Phys. 5, 2089–2097.
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
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 2–5 nm, spherical and monodispersed intracellular mercury nanoparticles. The remediated mercury trapped in the form of nanoparticles 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 characterized by high resolution transmission electron microscopy, energy dispersive X-ray analysis, powder Xray diffraction and atomic force microscopy. The strain can be exploited for metal bioaccumulation from environmental effluent and developing a green process for nanoparticles biosynthesis. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Nanoparticles are finding wide range of applications in biomedical 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 exchange, 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-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 ⇑ Corresponding author. Tel.: +91 11 26596533; fax: +91 11 26581102. E-mail address: [email protected] (S.K. Khare). 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.12.040
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 mercury Hg0. Present work explores mercury bioremediation with simultaneous 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 microorganism that was isolated from soil was used in the present study (Gupta 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 medium 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.
A. Sinha, S.K. Khare / Bioresource Technology 102 (2011) 4281–4284
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 recorded at A660 nm using double beam UV visible spectrophotometer (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 culture 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 synthesis. 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 (Perkin Elmer MHS-15 Mercury/Hydride System, USA). The mercury was estimated in each samples using sodium tetrahydroborate according to the recommended conditions provided by the manufacturer (Perkin Elmer MHS-15 Mercury/Hydride System, users guide, 2000). Effect of different parameters viz. pH, incubation time and metal concentration on the growth, bioaccumulation and nanoparticles synthesis by Enterobacter sp. was studied. Cells were cultivated in culture media described previously, except 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 incorporating varying concentrations of HgCl2 in the culture media 5 mg L 1, 10 mg L 1 or 15 mg L 1.
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 temperature. 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.
2.4.2. X-ray photoelectron spectroscopy (XPS) XPS was carried out to check the oxidation state of the accumulated nanoparticles. Twenty mL of 96 h bacterial culture grown in 5 mg L 1 of HgCl2 was centrifuged at 14,000g 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 charging effect.
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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 bioaccumulation 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.
Concentration of mercury (mgL )
2.4. Characterization of mercury nanoparticle
A660
4282
0
0
0
24
48
72 Time (h)
96
120
144
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; , residual mercury concentration in culture media in absence of Enterobacter sp. cells.
A. Sinha, S.K. Khare / Bioresource Technology 102 (2011) 4281–4284
4283
3.2. Characterization of accumulated mercury nanoparticles
3.4. Recovery of mercury nanoparticles by cell sonication
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 characterized by HRTEM, XPS and XRD. The high resolution transmission electron micrograph (HRTEM) image provides further insight into the structure of the intracellularly synthesized mercury nanoparticles (Supplementary data Fig. S1b). The image exhibits lattice fringes with d-spacing of 0.327 nm, which is consistent with the 0.327 nm separation 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 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 presence 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 remediated 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 particles around the metal center could be mediated through reductases, followed by aggregation with other cellular proteins (Nair and Pradeep, 2002; Brown et al., 2000).
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 intracellular 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 (Supplementary 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 nanoparticles. Both the profiles of recovered nanoparticles and those present in intact cytoplasm were consistent and same.
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 nanoparticles were investigated. Number of particles and their monodispersibility 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 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 distribution 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 mercury 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 nanoparticle synthesis is previously reported in case of Verticellum luteoalbum (Gericke and Pinches, 2006).
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 environment 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 acknowledged. Author Arvind Sinha is grateful to University Grant Commission, 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, 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 in nanobiomedicine. 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 ion exchange resins adsorption. Wat. Res. 34, 2971–2978. 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, 293–346. David, G.F.X., Herbert, J., Wright, G.D.S., 1973. The ultrastructure of the pineal ganglion in the ferret. J. Anat. 115, 79–97. Devi, P.S.R., Kumar, S., Sudersan, M., 2006. Sorption of mercury on chemically synthesized 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. Mercury resistance determinants related to Tn21, Tn1696, and Tn5053 in Enterobacteria from 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-mercury nanoparticles in aqueous solution. J. Phys. Chem. B 104, 5056–5060.
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