3 Biotech

, 8:45 | Cite as

Isolation, characterization and exploring biotechnological potential of halophilic archaea from salterns of western India

  • Aparna SinghEmail author
  • Anil Kumar Singh
Original Article


Thirteen halophilic archaea were isolated from Kandla and Bhayander salt pans. These isolates were grouped into three different genera Halobacterium, Haloferax and Haloarcula based on morphological and biochemical characterization, polar lipid analysis, Amplified 16S rDNA restriction analysis (ARDRA) and 16S rDNA sequence analysis. Biochemical characterization suggested the ability of isolates to produce protease, amylase and poly-hydroxybutyrate (PHB) indicating their biotechnological potential. The isolates were further screened for the amount of extracellular protease produced. Halobacterium sp. SP1(1) showed significant protease production compared to other isolates. Protease producing ability of the isolate was influenced by several factors such as NaCl concentration, type of protein source, metal ions and surfactants, and presence of amino acid supplements in the production medium. Soybean flour, FeCl3 and dicotylsulfosuccinate were found to increase protease production by 2.36, 1.54 and 1.26 folds, respectively compared to production in basal medium. Effect of organic solvents used in paints (n-decane, n-undecane and n-dodecane) was also investigated on protease production by the isolate. Protease production by Halobacterium sp. SP1(1) was enhanced by 1.2 folds in presence of n-decane compared to control. Furthermore, the ability of isolate to hydrolyse fish protein was investigated using three different edible fishes (Pomfret, Flat fish and Seer fish) as sole protein source. Pomfret was found to be a good protein source for protease production by the isolate. These results revealed that Halobacterium sp. SP1(1) may have potential for paint-based antifouling coating preparations and fish sauce preparation by virtue of its extracellular protease.


ARDRA Fish protein Halobacterium Halophilic archaea Organic solvents Protease Salt pans 



The work was supported by Council of Scientific and Industrial Research (CSIR) grant (90(0038)/04/EMR-II), New Delhi, India. The standard strain Halobacterium salinarum was gifted by Dr. H. M. Sonawat, Tata Institute of Fundamental Research, Mumbai, India. Heartiest thanks to Prof. A. J. Desai, M. S. University of Baroda, India, for providing lab facility and guidance.

Compliance with ethical standards

Conflict of interest

Authors report no conflict of interest.


  1. Adinarayana K, Ellaiah P, Prasad DS (2003) Purification and partial characterization of thermostable serine alkaline protease from a newly isolated Bacillus subtilis PE-11. AAPS PharmSci Tech 4:1–9CrossRefGoogle Scholar
  2. Akolkar AV (2009) Isolation and characterization of halophilic archaea: Production characterization and application of extracellular protease from Halobacterium SP SP1 1. Ph. D thesis, The Maharaja Sayajirao University of Baroda, Vadodara, Gujarat, IndiaGoogle Scholar
  3. Akolkar AV, Deshpande GM, Raval KN, Durai D, Nerurkar AS, Desai AJ (2008) Organic solvent tolerance of Halobacterium sp. SP1 (1) and its extracellular protease. J Basic Microbiol 48:421–425CrossRefGoogle Scholar
  4. Akolkar AV, Durai D, Desai AJ (2010) Halobacterium sp. SP1 (1) as a starter culture for accelerating fish sauce fermentation. J Appl Microbiol 109:44–53Google Scholar
  5. Balaban N, Gabdrakhmanova L, Sharipova M, Sokolova E, Malikova L, Mardanova A, Rudenskaya G, Leshchinskaya I (2004) Selection of cultivation medium for production of late stationary phase serine proteinases from Bacillus intermedius. J Basic Microbiol 44:415–423CrossRefGoogle Scholar
  6. Banerjee R, Bhattacharya BC (1992) Extracellular alkaline protease of newly isolate Rhizopus oryzae. Biotechnol Lett 14:301–304CrossRefGoogle Scholar
  7. Basinger GW, Oliver JD (1979) Inhibition of Halobacterium cutirubrum lipid biosynthesis by bacitracin. J Gen Microbiol 111:223–227CrossRefGoogle Scholar
  8. Capiralla H, Hiroi T, Hirokawa T, Maeda S (2002) Purification and characterization of a hydrophobic amino acid-specific endopeptidase from Halobacterium halobium S9 with potential application in debittering of protein hydrolysates. Process Biochem 38:571–579CrossRefGoogle Scholar
  9. D’Alessandro CP, De Castro RE, Giménez MI, Paggi RA (2007) Effect of nutritional conditions on extracellular protease production by haloalkaliphilic archaeon Natrialba magadii. Lett Appl Microbiol 44:637–642CrossRefGoogle Scholar
  10. Dave SR, Desai HB (2006) Microbial diversity at marine salterns near Bhavnagar, Gujarat, India. Curr Sci 90:497–500Google Scholar
  11. Don TM, Chen CW, Chan TH (2006) Preparation and characterization of poly (hydroxyalkanoate) from the fermentation of Haloferax mediterranei. J Biomater Sci Polym Edn. 17:1425–1438CrossRefGoogle Scholar
  12. Dyall-Smith M (2008) The halohandbook: protocols for halobacterial genetics (Version 7 electronic publication)Google Scholar
  13. Elevi R, Assa P, Birbir M, Ogan A, Oren A (2004) Characterization of extremely halophilc archaea isolated from the Ayvalik salterns, Turkey. World J Microbiol Biotechnol 20:719–725CrossRefGoogle Scholar
  14. Esakkiraj P, Immanuel G, Sowmya SM, Iyapparaj P, Palavesam A (2009) Evaluation of proteases-producing ability of fish gut isolate Bacillus cereus for aqua feed. Food Bioprocess Technol 2:383–390CrossRefGoogle Scholar
  15. Goh F, Jeon YJ, Barrow K, Neilan BA, Burns BP (2011) Osmoadaptive strategies of the archaeon Halococcus hamelinensis isolated from a hypersaline stromatolite environment. Astrobiology 11:529–536CrossRefGoogle Scholar
  16. Grant WD, Larsen H (1989) Extremely halophilic archaeobacteria. In: Staley, Bryant, Pfennig and Holt (ed.) Bergy’s manual of systematic bacteriology, 1st edn. Vol 3, Williams & Wilkins Co, Baltimore, pp. 2216–2219Google Scholar
  17. Grifantini R, Sebastian S, Frigimelica E, Draghi M, Bartolini E et al (2003) Identification of iron-activated and -repressed Fur-dependent genes by transcriptome analysis of Neisseria meningitides group B. Proc Natl Acad Sci USA 100:9542–9547CrossRefGoogle Scholar
  18. Gupta R, Lanter JM, Woese CR (1983) Sequence of the 16S ribosomal RNA from Halobacterium volcanii, an archaebacterium. Science 221:656–659CrossRefGoogle Scholar
  19. Hampp N, Oesterhelt D (2008) Bacteriorhodopsin and its potential in technical applications. Protein Science Encyclopedia. Wiley-VCH Verlag GmbH & Co. KGaA WeinheimGoogle Scholar
  20. Haseltine C, Hill T, Montalvo-Rodriguez R, Kemper SK, Shand RF, Blum P (2001) Secreted euryarchaeal microhalocins kill hyperthermophilic Crenarchaea. J Bacteriol 183:287–291CrossRefGoogle Scholar
  21. Kamekura M, Sen Y, Holmes ML, Dyall-Smith ML (1992) Molecular cloning and sequencing of the gene for a halophilic alkaline serine protease (halolysin) from an unidentified halophilic Archaea strain (172P1) and expression of the gene in Haloferax volcanii. J Bacteriol 174:736–742CrossRefGoogle Scholar
  22. Kerkar S (2004) Studies on bacteria of the dissimilatory reductive processes of the sulphur cycle from the salt pans of Goa. Ph.D, Thesis, Department of Marine Sciences, Goa University, Goa, IndiaGoogle Scholar
  23. Kim J, Dordick JS (1997) Unusual salt and solvent dependence of a protease from an extreme halophile. Biotech Bioeng 55:471–479CrossRefGoogle Scholar
  24. Kushwaha SC, Juez- Perez G, Kates M, Kushner DJ (1982) Survey of lipids of a new group of extremely halophilic bacteria from salt ponds in Spain. Can J Microbiol 28:1365–1372CrossRefGoogle Scholar
  25. Lillo JG, Rodriguez-Valera F (1990) Effect of culture conditions on poly-fi- hydroxybutyric acid production of Haloferax mediterranei. Appl Environ Microbiol 56:2517–2521Google Scholar
  26. Ma Y, Galinski EA, Grant WD, Oren A, Ventosa A (2010) Halophiles 2010: life in saline environments. Appl Environ Microbiol 76:6971–6981CrossRefGoogle Scholar
  27. Mancinelli RL, Hochstein LI (1986) The occurrence of denitrification in extremely halophilic bacteria. FEMS Microbiol Lett 35:55–58CrossRefGoogle Scholar
  28. Martínez-Espinosa RM, Zafrilla B, Camacho M, Bonete MJ (2007) Nitrate and nitrite removal from salted water by Haloferax mediterranei. Biocatal Biotransform 25:295–300CrossRefGoogle Scholar
  29. Maturrano L, Santos F, Rosselló-Mora R, Antón J (2006) Microbial diversity in Maras salterns, a hypersaline environment in the Peruvian Andes. Appl Environ Microbiol 72:3887–3895CrossRefGoogle Scholar
  30. Mutlu MB, Martínez-García M, Santos F, Peña A, Guven K, Antón J (2008) Prokaryotic diversity in Tuz Lake, a hypersaline environment in Inland Turkey. FEMS Microbiol Ecol 65:474–483CrossRefGoogle Scholar
  31. Oren A (2010) Industrial and environmental applications of halophilic microorganisms. Environ Technol 3:825–834CrossRefGoogle Scholar
  32. Oren A, Ventosa A, Grant WD (1997) Proposed minimal standards for description of new texa in the order Halobacteriales. Int J Sys Bacteriol 47:233–238CrossRefGoogle Scholar
  33. Patel R, Dodia M, Singh SP (2005) Extracellular alkaline protease from a newly isolated haloalkaliphilic Bacillus sp.: production and optimization. Process Biochem 40:3569–3575CrossRefGoogle Scholar
  34. Platas G, Meseguer I, Amils R (2002) Purification and biological characterization of halocin H1 from Haloferax mediterranei M2a. Int Microbiol 5:15–19CrossRefGoogle Scholar
  35. Raghavan TM, Furtado I (2000) Tolerance of an estuarine halophilic archaebacterium to crude oil and constituent hydrocarbons. Bull Environ Contam Toxicol 65:725–731CrossRefGoogle Scholar
  36. Reed CJ, Lewis H, Trejo E, Winston V, Evilia C (2013) Protein adaptations in archaeal extremophiles. Archaea 2013:1CrossRefGoogle Scholar
  37. Rohlf FJ (2009) NTSYSpc Numerical taxonomy and multivariate analysis system. Version 2.2. Getting Started Guide. Applied Biostatistics Inc., New YorkGoogle Scholar
  38. Shigematsu T, Fukushina J, Oyama M, Tsuda M, Kawamoto S, Okuda K (2001) Iron-mediated regulation of alkaline proteinase production in Pseudomonas aeruginosa. Microbiol Immunol 45:579–590CrossRefGoogle Scholar
  39. Suresh AK, Mody K, Jha B (2007) Bacterial exopolysaccharides—a perception. J Basic Microbiol 47:103–117CrossRefGoogle Scholar
  40. Thongthai C, Panbangred W, Choprasert C, Dhaveetiyanond S (1990) Protease activities in the traditional process of fish sauce fermentation. In: Reilly PJA, Parry RWH, Barile LE (eds) Post-harvest technology, preservation and quality of fish in Southeast Asia. Stockholm, International Foundation for Sciences, pp 61–65Google Scholar
  41. Tomlinson GA, Jahnke LL, Hochstein LI (1986) Halobacterium denitrificans sp nov, an extremely halophilic denitrifying bacterium. Int J SystBacteriol36:66–70Google Scholar
  42. Vaneechoutte M, De Beenhouwer H, Claeys G, Verschraegen G, De Rouck A, Paepe N, Elaichouni A, Portaels F (1993) Identification of mycobacterium species with amplified rDNA restriction analysis. J Clin Microbiol 31:2061–2065Google Scholar
  43. Vidyasagar M, Prakash S, Litchfield C, Sreeramulu K (2006) Purification and characterization of a thermostable, haloalkaliphilic extracellular serine protease from extreme halophilic archaeon Halogeometricum borinquense strain TSS101. Archaea 2:51–57CrossRefGoogle Scholar
  44. Wieland F, Lechner J, Sumper M (1982) The cell wall glycoprotein of halobacteium: structural, functional and biosynthetic aspects. Zbl Bakt Hyg J Abt Orig C3:161–170Google Scholar
  45. Winderickx J, Holsbeeks I, Lagatie O, Goits K, Thevelein J, Winde H (2003) In: yeast stress responses, topics in current genetics. Springer, Berlin, pp 307–386Google Scholar
  46. Xu X, Min W, Huang W (2005) Isolation and characterization of a novel strain of Natrinema containing a bop gene. J Zhejiang Univ Sci 6B:142–146CrossRefGoogle Scholar
  47. Yildiz E, Ozcan B, Caliskan M (2012) Isolation, characterization and phylogenetic analysis of Halophilic Archaea from a Salt Mine in Central Anatolia (Turkey). Pol J Microbiol 61:111Google Scholar
  48. Yongsawatdigul J, Rodtong S, Raksakulthai N (2007) Acceleration of Thai fish sauce fermentation using proteinases and bacterial starter cultures. J Food Sci 72:382–390CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2017

Authors and Affiliations

  1. 1.Department of Microbiology and Biotechnology Centre, Faculty of ScienceThe Maharaja Sayajirao University of BarodaVadodaraIndia
  2. 2.Department of BiotechnologyShree M. & N. Virani Science CollegeRajkotIndia

Personalised recommendations