, Volume 17, Issue 5, pp 821–831 | Cite as

Synthesis of silver nanoparticles using haloarchaeal isolate Halococcus salifodinae BK3

  • Pallavee Srivastava
  • Judith Bragança
  • Sutapa Roy Ramanan
  • Meenal KowshikEmail author
Original Paper


Numerous bacteria, fungi, yeasts and viruses have been exploited for biosynthesis of highly structured metal sulfide and metallic nanoparticles. Haloarchaea (salt-loving archaea) of the third domain of life Archaea, on the other hand have not yet been explored for nanoparticle synthesis. In this study, we report the intracellular synthesis of stable, mostly spherical silver nanoparticles (AgNPs) by the haloarchaeal isolate Halococcus salifodinae BK3. The culture on adaptation to silver nitrate exhibited growth kinetics similar to that of the control. NADH-dependent nitrate reductase was involved in silver tolerance, reduction, synthesis of AgNPs, and exhibited metal-dependent increase in enzyme activity. The AgNPs preparation was characterized using UV–visible spectroscopy, XRD, TEM and EDAX. The XRD analysis of the nanoparticles showed the characteristic Bragg peaks of face-centered cubic silver with crystallite domain size of 22 and 12 nm for AgNPs synthesized in NTYE and halophilic nitrate broth (HNB), respectively. The average particle size obtained from TEM analysis was 50.3 and 12 nm for AgNPs synthesized in NTYE and HNB, respectively. This is the first report on the synthesis of silver nanoparticles by haloarchaea.


Haloarchaea Biosynthesis Silver nanoparticles Nitrate reductase Metal tolerance Halococcus 



NaCl, tryptone, yeast extract


Silver nanoparticles


Metallic nanoparticles


Halophilic nitrate broth


X-ray diffraction


Transmission electron microscopy


Energy dispersive x-ray analysis


Selected area electron diffraction


Nitrate reductase


Face-centered cubic


Ethylene diamine tetra acetic acid


Phenyl methane sulfonyl fluoride




N-(1-Naphthyl) ethylene diamine hydrochloride


5,5′-Dithio-bis (2-nitrobenzoic acid)


Total thiol


Non-protein thiol


Protein-bound thiol


Cell-free extract


Zone of inhibition




Gamma glutamyl cysteine


bis-γ-Glutamyl cysteine reductase


Multiply-twinned particles



We thank Ministry of Earth Science (MoES), Government of India for their funding of the project MoES/11-MRDF/1/38/P/10-PC-III. We would like to thank Dr. Neha Hebalkar at ARCI, Hyderabad and the SAIF at IIT-Bombay for their help with TEM.

Supplementary material

792_2013_563_MOESM1_ESM.docx (17 kb)
Supplementary material 1 (DOCX 17 kb)
792_2013_563_MOESM2_ESM.tif (8.3 mb)
Supplemnetary Fig. S1 Effect of silver nitrate on the concentrations of total (T-SH), non-protein (NP-SH) and protein-bound (PB-SH) thiols in H. salifodinae BK3 exposed to 0.05 and 0.5 mM AgNO3. Control = 0 mM AgNO3. Values are mean ± SD for three experiments. (TIFF 8499 kb)
792_2013_563_MOESM3_ESM.tif (11 mb)
Supplementary Fig. S2 Effect of silver nitrate on growth profiles of H. salifodinae BK3 a in HNB without AgNO3 (control); b upon first exposure to AgNO3 by addition of 0.5 mM AgNO3 in HNB; c for cells adapted to AgNO3 upon addition of 0.5 mM AgNO3 in HNB. Values are mean ± SD (error bars) for three experiments. (TIFF 11254 kb)
792_2013_563_MOESM4_ESM.tif (7.8 mb)
Supplementary Fig. S3 UV–visible absorbance spectrum of the AgNPs synthesized by H. salifodinae BK3. a The spectra of AgNPs synthesized in NTYE (black) and in HNB (red); b Comparison of the UV–Visible profile of AgNPs prepared in HNB immediately after synthesis (blue) and after 6 months of storage (green). (TIFF 7995 kb)
792_2013_563_MOESM5_ESM.tif (7.8 mb)
Supplementary Fig. S4 X-ray diffraction pattern of AgNPs synthesized by H. salifodinae BK3 in a NTYE and b HNB. (TIFF 7995 kb)
792_2013_563_MOESM6_ESM.tif (1.2 mb)
Supplementary Fig. S5 Representative TEM micrographs showing triangular and disc like morphology of the AgNPs synthesized by H. salifodinae BK3 in the presence of 0.5 mM AgNO3. (TIFF 1256 kb)
792_2013_563_MOESM7_ESM.tif (269 kb)
Supplementary Fig. S6 EDAX spectrum of the AgNPs synthesized by H. salifodinae BK3 in a NTYE and b HNB. (TIFF 269 kb)


  1. Ahmad A, Mukherjee P, Senapati S, Mandal D, Khan MI, Kumar R (2003) Extracellular biosynthesis of silver nanoparticles using the fungus Fusarium oxysporum. Colloids Surf B 28:313–318CrossRefGoogle Scholar
  2. Avery SV (2001) Metal toxicity in yeast and the role of oxidative stress. Adv Appl Microbiol 49:111–142PubMedCrossRefGoogle Scholar
  3. Babu MMG, Sridhar J, Gunasekaran P (2011) Global transcriptome analysis of Bacillus cereus ATCC 14579 in response to silver nitrate stress. J Nanobiotechnol 9:49. doi: 10.1186/1477-3155-9-49 CrossRefGoogle Scholar
  4. Bai HJ, Zhang ZM, Guo Y, Yang GE (2009) Biosynthesis of cadmium sulfide nanoparticles by photosynthetic bacteria Rhodopseudomonas palustris. Colloids Surf B 70:142–146CrossRefGoogle Scholar
  5. Basavaraja S, Balaji SD, Lagashetty A, Rajasab AH, Venkataraman A (2008) Extracellular biosynthesis of silver nanoparticles using the fungus Fusarium semitectum. Mater Res Bull 43:1164–1170CrossRefGoogle Scholar
  6. Berney M, Weilenmann HU, Ihssen J, Bassin C, Egli T (2006) Specific growth rate determines the sensitivity of Escherichia coli to thermal, UVA, and solar disinfection. Appl Environ Microbiol 72:2586–2593PubMedCrossRefGoogle Scholar
  7. Borrelly GPM, Harrison MD, Robinson AK, Cox SG, Robinson NJ, Whitehall SK (2002) Surplus zinc is handled by Zym1 metallothionein and Zhf endoplasmic reticulum transporter in Schizosaccharomyces pombe. J Biol Chem 277:30394–30400PubMedCrossRefGoogle Scholar
  8. Breidt F, Romick TL, Fleming HF (1994) Rapid method for the determination of bacterial growth kinetics. J Rapid Methods Autom Microbiol 3:59–68CrossRefGoogle Scholar
  9. Catauro M, Raucci MG, De Gaetano F, Marotta A (2004) Antibacterial and bioactive silver-containing Na2O × CaO × SiO2 glass prepared by sol-gel method. J Mater Sci Mater Med 15:831–837PubMedCrossRefGoogle Scholar
  10. Danilcauk M, Lund A, Saldo J, Yamada H, Michalik J (2006) Conduction electron spin resonance of small silver particles. Spectrochimaca Acta Part A 63:189–191CrossRefGoogle Scholar
  11. Deepak V, Kalishwaralal K, Ram Kumar Pandian S, Gurunathan S (2011) An insight into the bacterial biogenesis of silver nanoparticles, industrial production and scale-up. In: Rai M, Durán N (eds) Metal nanoparticles in microbiology. Springer, Berlin, pp 17–35CrossRefGoogle Scholar
  12. Durán N, Marcato PD, Alves O, Souza G (2005) Mechanistic aspects of biosynthesis of silver nanoparticles by several Fusarium oxysporum strains. J Nanobiotechnol 3:8. doi: 10.1186/1477-3155-3-8 CrossRefGoogle Scholar
  13. Fahey RC (2001) Novel thiols of prokaryotes. Annu Rev Microbiol 55:333–356PubMedCrossRefGoogle Scholar
  14. Farrar WE (1985) Antibiotic resistance in developing countries. J Infect Dis 152:1103–1106PubMedCrossRefGoogle Scholar
  15. Feng QL, Wu J, Chen GQ, Cui FZ, Kim TN, Kim JO (2008) A mechanistic study of the antibacterial effect of silver ions on Escherichia coli and Staphylococcus aureus. J Biomed Mater Res 52:662–668CrossRefGoogle Scholar
  16. Furno F, Morley KS, Wong B, Sharp BL, Arnold PL, Howdle SM, Bayston R, Brown PD, Winship PD (2004) Silver nanoparticles and polymeric medical devices: a new approach to prevention of infection? J Antimicrob Chemother 54:1019–1024PubMedCrossRefGoogle Scholar
  17. Ge W, Zamri D, Mineyama H, Valix M (2011) Bioaccumulation of heavy metals on adapted Aspergillus foetidus. Adsorption 17:901–910CrossRefGoogle Scholar
  18. Gericke M, Pinches A (2006) Biological synthesis of metal nanoparticles. Hydrometallurgy 83:132–140CrossRefGoogle Scholar
  19. Guimaraes-Soares L, Pascoal C, Cassio F (2007) Effects of heavy metals on the production of thiol compounds by the aquatic fungi Fontanospora fusiramosa and Flagellospor acurta. Ecotoxicol Environ Saf 66:36–43PubMedCrossRefGoogle Scholar
  20. Hariharan H, Al-dhabi NA, Karuppiah P, Rajaram SK (2012) Microbial synthesis of selenium nanocomposite using Saccharomyces cerevisiae and its antimicrobial activity against pathogens causing nosocomial infection. Chalcogenide Lett 9:509–515Google Scholar
  21. Harley SM (1993) Use of a simple, colorimetric assay to demonstrate conditions for induction of nitrate reductase in plants. Am Biol Teach 55:161–164CrossRefGoogle Scholar
  22. Hayashi Y, Mutoh N (1994) Cadystin (phytochelatin) in fungi. In: Winkelmann G, Winge DR (eds) Metal ions in fungi. Marcel Dekker, New York, pp 311–337Google Scholar
  23. Hofmeister H (1999) Fivefold twinning in nanosized particles and nanocrystalline thin films—ubiquitous metastable structures. Mater Sci Forum 325:312–314Google Scholar
  24. Hutter E, Fendler JH (2004) Exploitation of localized surface plasmon resonance. Adv Mater 16:1685–1706CrossRefGoogle Scholar
  25. Hwang ET, Lee JH, Chae YJ, Kim YS, Kim BC, Sang B-I, Gu MB (2008) Analysis of the toxic mode of action of silver nanoparticles using stress-specific bioluminescent bacteria. Small 4:746–750PubMedCrossRefGoogle Scholar
  26. Jianping X, Jim YL, Daniel ICW, Yen PT (2007) Identification of active biomolecules in the high-yield synthesis of single-crystalline gold nanoplates in algal solutions. Small 3:668–672Google Scholar
  27. Kalimuthu K, Suresh Babu R, Venkataraman D, Bilal M, Gurunathan S (2008) Biosynthesis of silver nanocrystals by Bacillus licheniformis. Colloids Surf B 65:150–153CrossRefGoogle Scholar
  28. Kapoor S, Lawless D, Kennepohl P, Meisel D, Serpone N (1994) Reduction and aggregation of silver ions in aqueous gelatine solutions. Langmuir 10:3018–3022CrossRefGoogle Scholar
  29. Kaur A, Pan M, Meislin M, Facciotti MT, El-Gewely R, Baliga NS (2006) A systems view of haloarchaeal strategies to with stand stress from transition metals. Genome Res 16:841–854PubMedCrossRefGoogle Scholar
  30. Kim JS, Kuk E, Yu KN, Kim JH, Park SJ, Lee HJ, Kim SH, Park YK, Park YH, Hwang CY, Kim YK, Lee YS, Jeong DH, Cho MH (2007) Antimicrobial effects of silver nanoparticles. Nanomedicine 3:95–101PubMedCrossRefGoogle Scholar
  31. Kim SH, Lee HS, Ryu DS, Choi SJ, Lee DS (2011) Antibacterial activity of silver-nanoparticles against Staphylococcus aureus and Escherichia coli. Korean J Microbiol Biotechnol 39:77–85Google Scholar
  32. Kneer R, Kutchan TM, Hochberger A, Zenk MH (1992) Saccharomyces cerevisiae and Neurospora crassa contain heavy metal sequestering phytochelatin. Arch Microbiol 157:305–310PubMedCrossRefGoogle Scholar
  33. Kowshik M, Ashtaputre S, Kharrazi S, Vogel W, Urban J, Kulkarni SK, Paknikar KM (2003) Extracellular synthesis of silver nanoparticles by a silver-tolerant yeast strain MKY3. Nanotechnology 14:95–100CrossRefGoogle Scholar
  34. Lengke M, Fleet M, Southam G (2006) Biosynthesis of silver nanoparticles by filamentous cyanobacteria from a silver (I) nitrate complex. Langmuir 10:1021–1030Google Scholar
  35. Li Y, Duan X, Qian Y, Yang L, Liao H (1999) Nanocrystalline silver particles: synthesis, agglomeration, and sputtering induced by electron beam. J Colloid Interface Sci 209:347–349PubMedCrossRefGoogle Scholar
  36. Malki L, Yanku M, Borovok I, Cohen G, Mevarech M, Aharonowitz Y (2009) Identification and characterization of gshA, a gene encoding the glutamate-cysteine ligase in the halophilicarchaeon Haloferax volcanii. J Bacteriol 191:5196–5204PubMedCrossRefGoogle Scholar
  37. Mallick K, Witcomb MJ, Scurell MS (2004) Polymer stabilized silver nanoparticles: a photochemical synthesis route. J Mater Sci 39:4459–4463CrossRefGoogle Scholar
  38. Mani K, Salgaonkar BB, Braganca JM (2012) Culturable halophilic archaea at the initial and crystallization stages of salt production in a natural solar saltern of Goa, India. Aquat Biosyst 8:15. doi: 10.1186/2046-9063-8-15 PubMedCrossRefGoogle Scholar
  39. Marks LD (1994) Experimental studies of small particle structures. Rep Prog Phys 57:603–649CrossRefGoogle Scholar
  40. Matsumura Y, Yoshikata K, Kunisaki S, Tsuchido T (2003) Mode of bacterial action of silver zeolite and its comparison with that of silver nitrate. Appl Environ Microbiol 69:4278–4281PubMedCrossRefGoogle Scholar
  41. Mie G (1908) Beiträge zur Optik trüber Medien, speziell kolloidaler Metallösungen.Ann Phys 25:377-445 American translation at
  42. Miersch J, Tschimedbalshir M, Barlocher F, Grams Y, Pierau B, Schierhorn A, Krauss GJ (2001) Heavy metals and thiol compounds in Mucor racemosus and Articulospora tetracladia. Mycol Res 105:883–889CrossRefGoogle Scholar
  43. Mokhtari N, Daneshpajouh S, Atashdehghan R, Seyedbagheri S, Abdi K, Sarkar S, Minaian S, Shahverdi HR, Shahverdi AR (2008) Biological synthesis of very small silver nanoparticles by culture supernatant of Klebsiella pneumonia: the effects of visible-light irradiation and the liquid mixing process. Mater Res Bull 44:1415–1421CrossRefGoogle Scholar
  44. Monroe S, Polk R (2000) Antimicrobial use and bacterial resistance. Curr Opin Microbiol 3:496–501PubMedCrossRefGoogle Scholar
  45. Morones JB, Elechiguerra JL, Camacho A, Holt K, Kouri JB, Ramírez JP, Yacaman MJ (2005) The bactericidal effect of silver nanoparticles. Nanotechnology 16:2346–2353PubMedCrossRefGoogle Scholar
  46. Mulvaney P (1996) Surface plasmon spectroscopy of nanosized metal particles. Langmuir 12:788–800CrossRefGoogle Scholar
  47. Münger K, Germann UA, Lerch K (1987) The Neurospora crassa metallothionein gene. Regulation of expression and chromosomal location. J Biol Chem 262:7363–7367PubMedGoogle Scholar
  48. Nepijko SA, Hofmeister H, Sack-Kongehl H, Schlogl R (2000) Multiply twinned particles beyond the icosahedron. J Cryst Growth 213:129–134CrossRefGoogle Scholar
  49. Newton GL, Javor B (1985) Gamma-Glutamyl cysteine and thiosulfate are the major low-molecular-weight thiols in halobacteria. J Bacteriol 161:438–441PubMedGoogle Scholar
  50. Ng WV, Kennedy SP, Mahairas GG, Berquist B, Pan M, Shukla HD, Lasky SR, Baliga NS, Thorsson V, Sbrogna J, Swartzell S, Weir D, Hall J, Dahl TA, Welti R, Goo YA, Leithauser B, Keller K, Cruz R, Danson MJ, Hough DW, Maddocks DG, Jablonski PE, Krebs MP, Angevine CM, Dale H, Isenbarger TA, Peck RF, Pohlschroder M, Spudich JL, Jung KW, Alam M, Freitas T, Hou S, Daniels CJ, Dennis PP, Omer AD, Ebhardt H, Lowe TM, Liang P, Riley M, Hood L, DasSarma S (2000) Genome Sequence of Halobacterium species NRC-1. PNAS 97:12176–12181PubMedCrossRefGoogle Scholar
  51. Oren A (2008) Microbial life at high salt concentrations: phylogenetic and metabolic diversity. Saline Syst 4:2. doi: 10.1186/1746-1448-4-2 PubMedCrossRefGoogle Scholar
  52. Rai M, Yadav A, Gade A (2009) Silver nanoparticles as a new generation of antimicrobials. Biotechnol Adv 27:76–83PubMedCrossRefGoogle Scholar
  53. Ramezani F, Ramezani M, Talebi S (2010) Mechanistic aspects of biosynthesis of nanoparticles by several microbes. Nanocon 10:12–14Google Scholar
  54. Salamanca-Buentello F, Persad DL, Court EB, Martin DK, Daar AS, Singer PA (2005) Nanotechnology and the developing world. PLoS Med 2:e97PubMedCrossRefGoogle Scholar
  55. Sedlak J, Lindsay RH (1968) Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Elman’s reagent. Anal Biochem 25:192–205PubMedCrossRefGoogle Scholar
  56. Seshadri S, Saranya K, Kowshik M (2011) Green synthesis of lead sulfide nanoparticles by the lead resistant marine yeast, Rhodosporidium diobovatum. Biotechnol Prog 27:1464–1469PubMedCrossRefGoogle Scholar
  57. Seshadri S, Prakash A, Kowshik M (2012) Biosynthesis of silver nanoparticles by marine bacterium Idiomarina sp. PR58-8. Bull Mater Sci 35:1201–1205CrossRefGoogle Scholar
  58. Shahverdi AR, Minaian S, Shahverdi HR, Jamalifar H, Nohi A (2007) Rapid synthesis of silver nanoparticles using culture supernatants of Enterobacteria: a novel biological approach. Process Biochem 42:919–923CrossRefGoogle Scholar
  59. Shankar SS, Rai A, Ahmad A, Sastry M (2004) Biosynthesis of silver and gold nanoparticles from extracts of different parts of the geranium plant. Appl Nanosci 1:69–77Google Scholar
  60. Sharma VK, Yngard RA, Lin Y (2008) Silver nanoparticles: green synthesis and their antimicrobial activities. Adv Colloid Interface Sci 145:83–96PubMedCrossRefGoogle Scholar
  61. Sondi I, Salopek-Sondi B (2004) Silver nanoparticles as antimicrobial agent: a case study on E. coli as a model for Gram-negative bacteria. J Colloid Interface Sci 275:177–182PubMedCrossRefGoogle Scholar
  62. Speiser DM, Ortiz DF, Kreppel L, Scheel G, McDonald G, Ow DW (1992) Purine biosynthetic genes are required for cadmium tolerance in Schizosaccharomyces pombe. Mol Cell Biol 12:5301–5310PubMedGoogle Scholar
  63. Srivastava P, Kowshik M (2013) Mechanisms of metal resistance and homeostasis in haloarchaea. Archaea 16. doi: 10.1155/2013/732864
  64. Sundquist AR, Fahey RC (1989) The function of γ-glutamylcysteine and bis-γ-glutamylcysteine reductase in Halobacterium halobium. J Biol Chem 264:719–725PubMedGoogle Scholar
  65. Sweeney RY, Mao C, Gao X, Burt JL, Belcher AM, Georgiou G, Iverson BL (2004) Bacterial biosynthesis of cadmium sulphide nanocrystals. Chem Biol 11:1553–1559PubMedCrossRefGoogle Scholar
  66. Thakkar KN, Mhatre SS, Parikh YR (2010) Biological synthesis of metallic nanoparticles. Nanomed NBM 6:262–275Google Scholar
  67. Underwood S, Mulvaney P (1994) Effect of the solution refractive index on the color of gold colloids. Langmuir 10:3427–3430CrossRefGoogle Scholar
  68. Vaidyanathan R, Gopalram S, Kalishwaralal K, Deepak V, Pandian SR, Gurunathan S (2010) Enhanced silver nanoparticle synthesis by optimization of nitrate reductase activity. Colloids Surf B 75:335–341CrossRefGoogle Scholar
  69. Vo-Dinh T (2008) Nano biosensing using plasmonic nanoprobes. IEEE J Sel Topics Quantum Electron 14:198–205CrossRefGoogle Scholar
  70. Wang G, Kennedy SP, Fasiludeen S, Rensing C, DasSarma S (2004) Arsenic resistance in Halobacterium sp. strain NRC-1 examined by using an improved gene knockout system. J Bacteriol 186:3187–3194PubMedCrossRefGoogle Scholar
  71. Winge DR, Nielson KB, Gray WR, Hamer DH (1985) Yeast metallothionein sequence and metal binding properties. J Biol Chem 260:14464–14470PubMedGoogle Scholar
  72. Zafrilla B, Martínez-Espinosa RM, Alonso MA, Bonete MJ (2010) Biodiversity of Archaea and floral of two inland saltern ecosystems in the Alto Vinalopó Valley, Spain. Saline Syst 6:10. doi: 10.1186/1746-1448-6-10 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Japan 2013

Authors and Affiliations

  • Pallavee Srivastava
    • 1
  • Judith Bragança
    • 1
  • Sutapa Roy Ramanan
    • 2
  • Meenal Kowshik
    • 1
    Email author
  1. 1.Department of Biological SciencesBirla Institute of Technology and Science PilaniZuarinagarIndia
  2. 2.Chemical EngineeringBirla Institute of Technology and Science PilaniZuarinagarIndia

Personalised recommendations