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

Abstract

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.

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References

  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 4:1–9

    Article  Google 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, India

  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 48:421–425

    CAS  Article  Google 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 109:44–53

    CAS  Google 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 44:415–423

    CAS  Article  Google Scholar 

  6. Banerjee R, Bhattacharya BC (1992) Extracellular alkaline protease of newly isolate Rhizopus oryzae. Biotechnol Lett 14:301–304

    CAS  Article  Google Scholar 

  7. Basinger GW, Oliver JD (1979) Inhibition of Halobacterium cutirubrum lipid biosynthesis by bacitracin. J Gen Microbiol 111:223–227

    Article  Google 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 38:571–579

    CAS  Article  Google 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 44:637–642

    Article  Google Scholar 

  10. Dave SR, Desai HB (2006) Microbial diversity at marine salterns near Bhavnagar, Gujarat, India. Curr Sci 90:497–500

    Google 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. 17:1425–1438

    CAS  Article  Google Scholar 

  12. Dyall-Smith M (2008) The halohandbook: protocols for halobacterial genetics (Version 7 electronic publication)

  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 20:719–725

    CAS  Article  Google 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 2:383–390

    Article  Google 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 11:529–536

    CAS  Article  Google 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. 2216–2219

  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 100:9542–9547

    CAS  Article  Google Scholar 

  18. Gupta R, Lanter JM, Woese CR (1983) Sequence of the 16S ribosomal RNA from Halobacterium volcanii, an archaebacterium. Science 221:656–659

    CAS  Article  Google Scholar 

  19. Hampp N, Oesterhelt D (2008) Bacteriorhodopsin and its potential in technical applications. Protein Science Encyclopedia. Wiley-VCH Verlag GmbH & Co. KGaA Weinheim

  20. Haseltine C, Hill T, Montalvo-Rodriguez R, Kemper SK, Shand RF, Blum P (2001) Secreted euryarchaeal microhalocins kill hyperthermophilic Crenarchaea. J Bacteriol 183:287–291

    CAS  Article  Google 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 174:736–742

    CAS  Article  Google 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, India

  23. Kim J, Dordick JS (1997) Unusual salt and solvent dependence of a protease from an extreme halophile. Biotech Bioeng 55:471–479

    CAS  Article  Google 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 28:1365–1372

    CAS  Article  Google Scholar 

  25. Lillo JG, Rodriguez-Valera F (1990) Effect of culture conditions on poly-fi- hydroxybutyric acid production of Haloferax mediterranei. Appl Environ Microbiol 56:2517–2521

    Google Scholar 

  26. Ma Y, Galinski EA, Grant WD, Oren A, Ventosa A (2010) Halophiles 2010: life in saline environments. Appl Environ Microbiol 76:6971–6981

    CAS  Article  Google Scholar 

  27. Mancinelli RL, Hochstein LI (1986) The occurrence of denitrification in extremely halophilic bacteria. FEMS Microbiol Lett 35:55–58

    CAS  Article  Google Scholar 

  28. Martínez-Espinosa RM, Zafrilla B, Camacho M, Bonete MJ (2007) Nitrate and nitrite removal from salted water by Haloferax mediterranei. Biocatal Biotransform 25:295–300

    Article  Google 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 72:3887–3895

    CAS  Article  Google 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 65:474–483

    CAS  Article  Google Scholar 

  31. Oren A (2010) Industrial and environmental applications of halophilic microorganisms. Environ Technol 3:825–834

    Article  Google 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 47:233–238

    Article  Google Scholar 

  33. Patel R, Dodia M, Singh SP (2005) Extracellular alkaline protease from a newly isolated haloalkaliphilic Bacillus sp.: production and optimization. Process Biochem 40:3569–3575

    CAS  Article  Google Scholar 

  34. Platas G, Meseguer I, Amils R (2002) Purification and biological characterization of halocin H1 from Haloferax mediterranei M2a. Int Microbiol 5:15–19

    CAS  Article  Google Scholar 

  35. Raghavan TM, Furtado I (2000) Tolerance of an estuarine halophilic archaebacterium to crude oil and constituent hydrocarbons. Bull Environ Contam Toxicol 65:725–731

    CAS  Article  Google Scholar 

  36. Reed CJ, Lewis H, Trejo E, Winston V, Evilia C (2013) Protein adaptations in archaeal extremophiles. Archaea 2013:1

    Article  Google Scholar 

  37. Rohlf FJ (2009) NTSYSpc Numerical taxonomy and multivariate analysis system. Version 2.2. Getting Started Guide. Applied Biostatistics Inc., New York

  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 45:579–590

    CAS  Article  Google Scholar 

  39. Suresh AK, Mody K, Jha B (2007) Bacterial exopolysaccharides—a perception. J Basic Microbiol 47:103–117

    Article  Google 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 61–65

    Google Scholar 

  41. Tomlinson GA, Jahnke LL, Hochstein LI (1986) Halobacterium denitrificans sp nov, an extremely halophilic denitrifying bacterium. Int J SystBacteriol36:66–70

  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 31:2061–2065

    CAS  Google 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 2:51–57

    CAS  Article  Google 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 C3:161–170

    Google 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 307–386

    Google 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:142–146

    CAS  Article  Google 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 61:111

    CAS  Google Scholar 

  48. Yongsawatdigul J, Rodtong S, Raksakulthai N (2007) Acceleration of Thai fish sauce fermentation using proteinases and bacterial starter cultures. J Food Sci 72:382–390

    Article  Google Scholar 

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Acknowledgement

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.

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Correspondence to Aparna Singh.

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Singh, A., Singh, A.K. Isolation, characterization and exploring biotechnological potential of halophilic archaea from salterns of western India. 3 Biotech 8, 45 (2018). https://doi.org/10.1007/s13205-017-1072-3

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Keywords

  • ARDRA
  • Fish protein
  • Halobacterium
  • Halophilic archaea
  • Organic solvents
  • Protease
  • Salt pans