Functional Screening and Molecular Characterization of Halophilic and Halotolerant Bacteria by 16S rRNA Gene Sequence Analysis

  • Bijay Kumar BeheraEmail author
  • Priyanka Das
  • Jitendra Maharana
  • Dharmendra Kumar Meena
  • Tanmaya Kumar Sahu
  • A. R. Rao
  • Soumendranath Chatterjee
  • Bimal Prasanna Mohanty
  • Anil Prakash Sharma
Research Article


A total of 206 bacterial isolates were obtained from soil, water and sediment samples using three different culture media with a pH of 7.3 ± 0.2 and an incubation temperature of 37 °C. Of these isolates, 48 bacteria were selected for functional screening and molecular characterization. The functional screening for salt stress tolerance revealed that, 17 bacteria were identified as moderately halotolerant and 31 were halophilic, capable of growing at 5–15 % (w/v) and ≥15 % (w/v) of NaCl concentration respectively. The 16S rRNA gene sequence analysis and taxonomic identification of isolates were performed using Ribosomal Database Project Release-10 and SILVA SSU database. Based on the sequence length >1,300 bp, 40 bacteria were identified out of 48 isolates. Study showed that, Staphylococcus spp. was highly resistant to salt stress followed by Enterococcus faecalis, Vagococcus fluvialis and Vibrio parahaemolyticus. The phylogenetic analysis revealed that, 24 bacterial isolates belong to Firmicutes and 16 isolates come under Proteobacteria. These bacterial strains would be of great use for prospecting novel and candidate salt stress tolerant genes.


Halophilic and halotolerant bacteria 16S rRNA gene Salt stress tolerance Molecular characterization 



Authors are greatly indebted to Dr. S. Ayyappan, Secretary, DARE and DG, ICAR, New Delhi, Dr. T. Mohapatra, Director, CRRI, Cuttack, and Dr. N. K. Singh, National Professor, NRCPB, New Delhi for their encouragement and support. This work was supported financially by the World Bank funded NAIP Project of ICAR on, “Bioprospecting of genes and allele mining for Abiotic stress tolerance”. The laboratory assistance by Mr. Asim Kumar Jana and Sk. Rabiul are duly acknowledged.


  1. 1.
    Baati H, Amdouni R, Gharsallah N (2010) Isolation and characterization of moderately halophilic bacteria from Tunisian solar saltern. Curr Microbiol 60:157–161CrossRefPubMedGoogle Scholar
  2. 2.
    Margesin, Schinner F (2001) Potential of halotolerant and halophilic microorganisms for biotechnology. Extremophiles 5:73–83CrossRefPubMedGoogle Scholar
  3. 3.
    Caton TM, Witte LR, Ngyuen HD, Buchheim JA, Buchheim MA, Schneegurt MA (2004) Halotolerant aerobic heterotrophic bacteria from the great salt plains of Oklahoma. Microb Ecol 48:449–462CrossRefPubMedGoogle Scholar
  4. 4.
    Grant WD (2004) Life at low water activity. Philos Trans R Soc Lond B 359:1249–1267CrossRefGoogle Scholar
  5. 5.
    Jiang HC, Dong HL, Zhang GX, Yu BS, Chapman LR, Fields MW (2006) Microbial diversity in water and sediment of Lake Chaka, an Athalassohaline Lake in northwestern China. Appl Environ Microbiol 72:3832–3845PubMedCentralCrossRefPubMedGoogle Scholar
  6. 6.
    Tsiamis G, Katsaveli K, Ntougias S, Kyrpides N, Andersen G, Piceno Y, Bourtzis K (2008) Prokaryotic community profiles at different operational stages of a Greek solar saltern. Res Microbiol 159:609–627CrossRefPubMedGoogle Scholar
  7. 7.
    Xiang WL, Guo JH, Feng W, Huang M, Chen H, Zhao J, Zhang J, Yang ZR, Sun Q (2008) Community of extremely halophilic bacteria in historic Dagong brine well in southwestern China. World J Microbiol Biotechnol 24:2297–2305CrossRefGoogle Scholar
  8. 8.
    Jenkins JN, Parrott WL, McCarty JC Jr et al (1991) Field test of transgenic cottons containing a Bacillus thuringiensis gene. Exp Stn Tech Bull 174:6Google Scholar
  9. 9.
    Kushner DJ (1993) Growth and nutrition of halophilic bacteria. In: Vreeland RH, Hochstein LI (eds) The biology of halophilic bacteria. CRC Press Inc., Boca Raton, pp 87–89Google Scholar
  10. 10.
    Prakash O, Verma M, Sharma P (2007) Polyphasic approach of bacterial classification—an overview of recent advances. Indian J Microbiol 47:98–108PubMedCentralCrossRefPubMedGoogle Scholar
  11. 11.
    Mignard S, Flandrois JP (2006) 16S rRNA sequencing in routine bacterial identification: a 30-month experiment. J Microbiol Methods 67:574–581CrossRefPubMedGoogle Scholar
  12. 12.
    MacFaddin JF (1985) Media for isolation-cultivation-identification-maintenance of medical bacteria, 1st edn. Williams & Wilkins Co., BaltimoreGoogle Scholar
  13. 13.
    Forbes BA, Sahm DF, Weissfeld AS (1998) Bailey and Scotts diagnostic microbiology, 10th edn. Mosby Inc., St. LouisGoogle Scholar
  14. 14.
    ZoBell CE (1941) Studies on marine bacteria. I. The cultural requirements of heterotrophic aerobes. J Mar Res 4:42–75Google Scholar
  15. 15.
    Macleod RA (1965) The question of the existence of specific marine bacteria. Bacteriol Rev 29:9–24PubMedCentralPubMedGoogle Scholar
  16. 16.
    Tang J, Zheng A, Bromfield ESP (2011) 16S rRNA gene sequence analysis of halophilic and halotolerant bacteria isolated from a hypersaline pond in Sichuan, China. Ann Microbiol 61:375–381CrossRefGoogle Scholar
  17. 17.
    Caton TM, Witte LR, Ngyuen HD, Buchheim JA, Buchheim MA, Schneegurt MA (2004) Halotolerant aerobic heterotrophic bacteria from the great salt plains of Oklahoma. Microb Ecol 48:449–462CrossRefPubMedGoogle Scholar
  18. 18.
    Sambrook J, Russel DW (2001) Molecular cloning: a laboratory manual, 3rd edn. CSH Laboratory Press, Cold Spring Harbor, p 5.62Google Scholar
  19. 19.
    Edwards U, Rogall T, Blöcker H (1989) Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA. Nucleic Acids Res 17:7843–7853PubMedCentralCrossRefPubMedGoogle Scholar
  20. 20.
    Altschul SF, Gish W, Miller W (1990) Basic local alignment search tool. J Mol Biol 215:403–410CrossRefPubMedGoogle Scholar
  21. 21.
    Larkin MA, Blackshields G, Brown NP (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23:2947–2948CrossRefPubMedGoogle Scholar
  22. 22.
    Hillis DM, Bull J (1993) An empirical test of bootstrapping as a method for assessing confidence in phylogenetic analysis. Syst Biol 42:182–192CrossRefGoogle Scholar
  23. 23.
    Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111–120CrossRefPubMedGoogle Scholar
  24. 24.
    Tamura K, Peterson D, Peterson N (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739PubMedCentralCrossRefPubMedGoogle Scholar
  25. 25.
    Fredricks DN, Relman DA (1996) Sequence-based identification of microbial pathogens: a reconsideration of Koch’s postulates. Clin Microbiol Rev 9:18–33PubMedGoogle Scholar
  26. 26.
    Shivanand P, Mugeraya G (2011) Halophilic bacteria and their compatible solutes—osmoregulation and potential applications. Curr Sci 100:1516–1521Google Scholar
  27. 27.
    den Besten HM, Mols M, Moezelaar R (2009) Phenotypic and transcriptomic analyses of mildly and severely salt-stressed Bacillus cereus ATCC 14579 cells. Appl Environ Microbiol 75:4111–4119CrossRefGoogle Scholar
  28. 28.
    Tsai M, Ohniwa RL, Kato Y (2011) Staphylococcus aureus requires cardiolipin for survival under conditions of high salinity. BMC Microbiol 11:13PubMedCentralCrossRefPubMedGoogle Scholar
  29. 29.
    Khare E, Singh S, Maheshwari DK (2011) Suppression of charcoal rot of chickpea by fluorescent Pseudomonas under saline stress condition. Curr Microbiol 62:1548–1553CrossRefPubMedGoogle Scholar
  30. 30.
    Yamaguchi T, Blumwald E (2005) Developing salt-tolerant crop plants: challenges and opportunities. Trends Plant Sci 10:615–620CrossRefPubMedGoogle Scholar

Copyright information

© The National Academy of Sciences, India 2014

Authors and Affiliations

  • Bijay Kumar Behera
    • 1
    Email author
  • Priyanka Das
    • 1
    • 3
  • Jitendra Maharana
    • 1
  • Dharmendra Kumar Meena
    • 1
  • Tanmaya Kumar Sahu
    • 2
  • A. R. Rao
    • 2
  • Soumendranath Chatterjee
    • 3
  • Bimal Prasanna Mohanty
    • 4
  • Anil Prakash Sharma
    • 5
  1. 1.Biotechnology LaboratoryCentral Inland Fisheries Research InstituteKolkataIndia
  2. 2.Indian Agricultural Statistics Research InstituteNew DelhiIndia
  3. 3.Parasitology and Microbiology LaboratoryUniversity of BurdwanBurdwanIndia
  4. 4.Biochemistry LaboratoryCentral Inland Fisheries Research InstituteKolkataIndia
  5. 5.Central Inland Fisheries Research InstituteKolkataIndia

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