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Characterization of extremely halophilic archaeal isolates from Indian salt pans and their screening for production of hydrolytic enzymes

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Abstract

Halophilic archaea are multi-stress resistant organisms and their enzymes are of special interest as they are generally stable and functional under extreme conditions of temperature and low water activity. The search for novel extremozymes is an ongoing one because of their superior functionality in extreme conditions encountered in various industries. In this study, extremely halophilic archaea were isolated from two salterns (Marakkanam and Vedaranyam) in Tamil Nadu and from three salterns (Sinquetim, Siridao and Ribandar) in Goa, India. All isolates were Gram negative and their pigmentation ranged from light pink to bright orange. Characterization of the ten halophilic archaeal isolates was carried out by morphological, biochemical and molecular techniques. They spanned 6 different genera; Haloferax, Halorubrum, Halococcus, Haloarcula, Halogeometricum, and Haloterrigena. These extremely halophilic archaeal strains were screened for production of hydrolytic enzymes like amylase, esterase, lipase, protease, pectinase and cellulase. Amylase production by Halogeometricum sp. is being reported for the first time. All isolates showed at least one enzyme activity. Halococcus sp., Haloarcula sp., and Haloferax sp. were capable of producing three extracellular enzymes each. Cellulolytic activity was not observed in any of the isolates. Since these enzymes are inherently salt tolerant, they are very promising from the industrial point of view.

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References

  • Akmoussi-Toumi S, Khemili-Talbi S, Ferioune I, Kebbouche-Gana S (2018) Purification and characterization of an organic solvent-tolerant and detergent-stable lipase from Haloferax mediterranei CNCMM 50101. Int J Biol Macromol 116:817–830. https://doi.org/10.1016/j.ijbiomac.2018.05.087

    Article  CAS  Google Scholar 

  • Alsafadi D, Paradisi F (2013) Effect of organic solvents on the activity and stability of halophilic alcohol dehydrogenase (ADH2) from Haloferax volcanii. Extremophiles 17(1):115–122

    Article  CAS  Google Scholar 

  • Anderson I, Scheuner C, Göker M, Mavromatis K, Hooper SD, Porat I, Klenk HP, Ivanova N, Kyrpides N (2011) Novel insights into the diversity of catabolic metabolism from ten haloarchaeal genomes. PLoS One 6(5):e20237

    Article  CAS  Google Scholar 

  • Antunes A, Taborda M, Huber R, Moissl C, Nobre MF, da Costa MS (2008) Halorhabdus tiamatea sp. nov., a non-pigmented, extremely halophilic archaeon from a deep-sea, hypersaline anoxic basin of the Red Sea, and emended description of the genus Halorhabdus. Int J Syst Evolut Microbiol 58(1):215–220

    Article  CAS  Google Scholar 

  • Balkrishna SB (2015) Synthesis of polyhydroxyalkanoates by halophilic archaea and bacteria and their osmoadaptation. Doctoral dissertation, Birla Institute of Technology and Science, Pilani)

  • Bardavid RE, Oren A (2008) Dihydroxyacetone metabolism in Salinibacter ruber and in Haloquadratum walsbyi. Extremophiles 12(1):125–131

    Article  CAS  Google Scholar 

  • BBC Research (2012) In Report BIO030G—Global markets for enzymes in industrial applications. https://www.bccresearch.com/market-research/biotechnology/enzymes-industrial-applications-markets-bio030g.html

  • Birbir M, Calli B, Mertoglu B, Bardavid RE, Oren A, Ogmen MN, Ogan A (2007) Extremely halophilic Archaea from Tuz Lake, Turkey, and the adjacent Kaldirim and Kayacik salterns. World J Microbiol Biotechnol 23(3):309–316

    Article  CAS  Google Scholar 

  • Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37(8):911–917

    Article  CAS  Google Scholar 

  • Burns DG, Janssen PH, Itoh T, Kamekura M, Li Z, Jensen G, Dyall-Smith ML (2007) Haloquadratum walsbyi gen. nov., sp. nov., the square haloarchaeon of Walsby, isolated from saltern crystallizers in Australia and Spain. Int J Syst Evolut Microbiol 57(2):387–392

    Article  CAS  Google Scholar 

  • Camacho RM, Mateos JC, González-Reynoso O, Prado LA, Córdova J (2009) Production and characterization of esterase and lipase from Haloarcula marismortui. J Ind Microbiol Biotechnol 36(7):901–909

    Article  CAS  Google Scholar 

  • Chu X, He H, Guo C, Sun B (2008) Identification of two novel esterases from a marine metagenomic library derived from South China Sea. Appl Microbiol Biotechnol 80(4):615–625

    Article  CAS  Google Scholar 

  • Cui HL, Tohty D, Zhou PJ, Liu SJ (2006) Halorubrum lipolyticum sp. nov. and Halorubrum aidingense sp. nov., isolated from two salt lakes in Xin-Jiang, China. Int J Syst Evolut Microbiol 56(7):1631–1634

    Article  CAS  Google Scholar 

  • Cui HL, Lin ZY, Dong Y, Zhou PJ, Liu SJ (2007) Halorubrum litoreum sp. nov., an extremely halophilic archaeon from a solar saltern. Int J Syst Evolut Microbiol 57(10):2204–2206

    Article  CAS  Google Scholar 

  • De Rosa M, Trincone A, Nicolaus B, Gambacorta A (1991) Archaebacteria: lipids, membrane structures, and adaptation to environmental stresses. Life under extreme conditions. Springer, Berlin, Heidelberg, pp 61–87

    Chapter  Google Scholar 

  • Dussault HP (1955) An improved technique for staining red halophilic bacteria. J Bacteriol 70(4):484

    CAS  Google Scholar 

  • Dyall-Smith M (2008) The halohandbook: protocols for halobacterial genetics. http://www.haloarchaea.com/resources/halohandbook/index.html. Accessed 31 Aug 2018

  • Elevi R, Assa P, Birbir M, Ogan A, Oren A (2004) Characterization of extremely halophilic Archaea isolated from the Ayvalik Saltern, Turkey. World J Microbiol Biotechnol 20(7):719–725

    Article  CAS  Google Scholar 

  • Elferink MGL, De Wit JG, Demel R, Driessen AJM, Konings WN (1992) Functional reconstitution of membrane proteins in monolayer liposomes from bipolar lipids of Sulfolobus acidocaldarius. J Biol Chem 267:1375–1381

    CAS  Google Scholar 

  • Fang Z, Li J, Wang Q, Fang W, Peng H, Zhang X, Xiao Y (2014) A novel esterase from a marine metagenomic library exhibiting salt tolerance ability. World J Microbiol Biotechnol 24:771–780

    Article  CAS  Google Scholar 

  • Gattinger A, Günthner A, Schloter M, Munch JC (2003) Characterization of archaea in soils by polar lipid analysis. Eng Life Sci 23(1):21–28

    CAS  Google Scholar 

  • Goh F, Leuko S, Allen MA, Bowman JP, Kamekura M, Neilan BA, Burns BP (2006) Halococcus hamelinensis sp. nov., a novel halophilic archaeon isolated from stromatolites in Shark Bay, Australia. Int J Syst Evolut Microbiol 56(6):1323–1329

    Article  CAS  Google Scholar 

  • Grant WD (2004) Life at low water activity. Philos Trans R Soc Lond B Biol Sci 359(1448):1249–1267

    Article  CAS  Google Scholar 

  • Kakhki AM, Amoozegar MA, Khaledi EM (2011) Diversity of hydrolytic enzymes in haloarchaeal strains isolated from salt lake. Int J Environ Sci Technol 8(4):705–714

    Article  CAS  Google Scholar 

  • Kamekura M (1999) Diversity of members of the family Halobacteriaceae. Microbiol Biogeochem Hypersaline Environ 13:26

    Google Scholar 

  • Kamekura M, Kates M (1999) Structural diversity of membrane lipids in members of Halobacteriaceae. Biosci Biotechnol Biochem 63(6):969–972

    Article  CAS  Google Scholar 

  • Karan R, Capes MD, Das Sarma S (2012) Function and biotechnology of extremophilic enzymes in low water activity. Aquat Biosyst 8:4

    Article  CAS  Google Scholar 

  • Karray F, Abdallah MB, Kallel N, Hamza M, Fakhfakh M, Sayadi S (2018) Extracellular hydrolytic enzymes produced by halophilic bacteria and archaea isolated from hypersaline lake. Molecular Biology Reports 45(5):1297–1309

    Article  CAS  Google Scholar 

  • Kates M (1977) The phytanyl ether-linked polar lipids and isoprenoid neutral lipids of extremely halophilic bacteria. Prog Chem Fats Other Lipids 15(4):301–342

    Article  Google Scholar 

  • Kates M (1993) Membrane lipids of archaea. New comprehensive biochemistry, vol 26. Elsevier, Amsterdam, pp 261–295

    Google Scholar 

  • Kaur S, Purohit MK (2012) Rainfall Statistics of India, Indian Meteorological Department, Ministry of earth sciences. Report number: ESSO/IMD/HS/R.F. REP/02 (2013)/16

  • Konings WN, Albers SV, Koning S, Driessen AJ (2002) The cell membrane plays a crucial role in survival of bacteria and archaea in extreme environments. Antonie Van Leeuwenhoek 81(1–4):61–72

    Article  CAS  Google Scholar 

  • Kumar S, Karan R, Kapoor S, Singh SP, Khare SK (2012) Screening and isolation of halophilic bacteria producing industrially important enzymes. Braz J Microbiol 43(4):1595–1603

    Article  CAS  Google Scholar 

  • Legat A, Denner E, Dornmayr-Pfaffenhuemer M, Pfeiffer P, Knopf B, Claus H, Stan-Lotter H (2013) Properties of Halococcus salifodinae, an isolate from Permian rock salt deposits, compared with halococci from surface waters. Life 3(1):244–259

    Article  Google Scholar 

  • Levin L, Herrmann C, Papinutti VL (2008) Optimization of lignocellulolytic enzyme production by the white-rot fungus Trametes trogii in solid-state fermentation using response surface methodology. Biochem Eng J 39(1):207–214

    Article  CAS  Google Scholar 

  • Li X, Yu HY (2014) Characterization of an organic solvent-tolerant lipase from Haloarcula sp. G41 and its application for biodiesel production. Folia Microbiol 59(6):455–463

    Article  CAS  Google Scholar 

  • Litchfield CD, Irby A, Kis-Papo T, Oren A (2000) Comparisons of the polar lipid and pigment profiles of two solar salterns located in Newark, California, USA, and Eilat, Israel. Extremophiles 4(5):259–265

    Article  CAS  Google Scholar 

  • Liu XD, Xu Y (2008) A novel raw starch digesting α-amylase from a newly isolated Bacillus sp. YX-1: purification and characterization. Bioresour Technol 99(10):4315–4320

    Article  CAS  Google Scholar 

  • Madern D, Ebel C, Zaccai G (2000) Halophilic adaptation of enzymes. Extremophiles 4(2):91–98

    Article  CAS  Google Scholar 

  • Mahadik ND, Puntambekar US, Bastawde KB, Khire JM, Gokhale DV (2002) Production of acidic lipase by Aspergillus niger in solid state fermentation. Process Biochem 38(5):715–721

    Article  CAS  Google Scholar 

  • 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(1):15

    Article  CAS  Google Scholar 

  • Markets and Markets (2014) In Report Code: FB 2277 industrial enzymes market by Types (Carbohydrase, Protease, Lipase), applications (food & beverages, cleaning agents, bio-fuel, animal feed), & Geography—Global Trends & Forecasts to 2018

  • Markets and Markets (2016) In Report Code: FB 2277 industrial enzymes market by type (Amylases, Cellulases, Proteases, Lipases, and Phytases), application (food and beverages, cleaning agents, and animal feed), source (microorganism, plant, and animal), and Region—Global Forecast to 2022

  • Martin DD, Ciulla RA, Roberts MF (1999) Osmoadaptation in archaea. Appl Environ Microbiol 65(5):1815–1825

    CAS  Google Scholar 

  • Minegishi H, Echigo A, Nagaoka S, Kamekura M, Usami R (2010) Halarchaeum acidiphilum gen. nov., sp. nov., a moderately acidophilic haloarchaeon isolated from commercial solar salt. Int J Syst Evolut Microbiol 60(11):2513–2516

    Article  CAS  Google Scholar 

  • Moldoveanu N, Kates M, Montero CG, Ventosa A (1990) Polar lipids of non-alkaliphilic Halococci. Biochim Biophys Acta (BBA) Lipids Lipid Metab 1046(2):127–135

    Article  CAS  Google Scholar 

  • Montalvo-Rodriguez RAFAEL, Vreeland RH, Oren A, Kessel M, Betancourt C, López-Garriga JUAN (1998) Halogeometricum borinquense gen. nov., sp. nov., a novel halophilic archaeon from Puerto Rico. Int J Syst Evolut Microbiol 48(4):1305–1312

    CAS  Google Scholar 

  • Moreno ML, Mellado E, Garcia MT, Ventosa A (2007) Diversity of extreme halophiles producing hydrolytic enzymes in hypersaline habitats. Halophiles-2007 booklet, 59–60

  • Moshfegh M, Shahverdi AR, Zarrini G, Faramarzi MA (2013) Biochemical characterization of an extracellular polyextremophilic α-amylase from the halophilic archaeon Halorubrum xinjiangense. Extremophiles 17:1–11

    Article  CAS  Google Scholar 

  • Müller-Santos M, de Souza EM, Pedrosa FDO, Mitchell DA, Longhi S, Carrière F, Krieger N (2009) First evidence for the salt-dependent folding and activity of an esterase from the halophilic archaea Haloarcula marismortui. Biochim Biophys Acta (BBA) Mol Cell Biol Lipids 1791(8):719–729

    Article  CAS  Google Scholar 

  • Nayek A, Gupta PSS, Banerjee S, Mondal B, Bandyopadhyay AK (2014) Salt-bridge energetics in halophilic proteins. PLoS One 9(4):e93862

    Article  Google Scholar 

  • Oren A (2010) Industrial and environmental applications of halophilic microorganisms. Environ Technol 31(8–9):825–834

    Article  CAS  Google Scholar 

  • Oren A, Duker S, Ritter S (1996) The polar lipid composition of Walsby’s square bacterium. FEMS Microbiol Lett 138(2–3):135–140

    Article  CAS  Google Scholar 

  • Promchai R, Boonchalearn A, Visessanguan W, Luxananil P (2018) Rapid production of extracellular thermostable alkaline halophilic protease originating from an extreme haloarchaeon, Halobacterium salinarum by recombinant Bacillus subtilis. Biocatal Agric Biotechnol 15:192–198

    Article  Google Scholar 

  • Rathod BN, Bhatt HH, Upasani VN (2016) Extracellular Hydrolases producing Haloarchaea from Marine Salterns at Okhamadhi, Gujarat, India. Int J Curr Microbiol App Sci 5(11):51–64

    Article  CAS  Google Scholar 

  • Reed CJ, Lewis H, Trejo E, Winston V, Evilia C (2013) Protein adaptations in archaeal extremophiles. Archaea. https://doi.org/10.1155/2013/373275

    Article  Google Scholar 

  • Rodrigo-Baños M, Garbayo I, Vílchez C, Bonete MJ, Martínez-Espinosa RM (2015) Carotenoids from haloarchaea and their potential in biotechnology. Mar Drugs 13(9):5508–5532

    Article  CAS  Google Scholar 

  • Rohban R, Amoozegar MA, Ventosa A (2009) Screening and isolation of halophilic bacteria producing extracellular hydrolyses from Howz Soltan Lake, Iran. J Ind Microbiol Biot 36(3):333–340

    Article  CAS  Google Scholar 

  • Sánchez-Porro C, Martin S, Mellado E, Ventosa A (2003) Diversity of moderately halophilic bacteria producing extracellular hydrolytic enzymes. J Appl Microbiol 94(2):295–300

    Article  Google Scholar 

  • Shafiei M, Ziaee AA, Amoozegar MA (2010) Purification and biochemical characterization of a novel SDS and surfactant stable, raw starch digesting, and halophilic α-amylase from a moderately halophilic bacterium, Nesterenkonia sp. strain F. Process Biochem 45(5):694–699

    Article  CAS  Google Scholar 

  • Singh A, Singh AK (2017) Haloarchaea: worth exploring for their biotechnological potential. Biotech Lett 39(12):1793–1800

    Article  CAS  Google Scholar 

  • Sinha R, Khare SK (2012) Characterization of detergent compatible protease of a halophilic Bacillus sp. EMB9: differential role of metal ions in stability and activity. Bioresour Technol 145:357–361

    Article  CAS  Google Scholar 

  • Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28(10):2731–2739

    Article  CAS  Google Scholar 

  • Thompson DH, Wong KF, Humphry-Baker R, Wheeler JJ, Kim JM, Rananavare SB (1992) Tetraether bolaform amphiphiles as models of archaebacterial membrane lipids: raman spectroscopy, phosphorus-31 NMR, x-ray scattering, and electron microscopy. J Am Chem Soc 114(23):9035–9042

    Article  CAS  Google Scholar 

  • Trigui H, Masmoudi S, Brochier-Armanet C, Maalej S, Dukan S (2011) Characterization of Halorubrum sfaxense sp. nov., a new halophilic archaeon isolated from the solar saltern of Sfax in Tunisia. Int J Microbiol. https://doi.org/10.1155/2011/240191

    Article  Google Scholar 

  • Ventosa A, Nieto JJ, Oren A (1998) Biology of moderately halophilic aerobic bacteria. Microbiol Mol Biol R 62(2):504–544

    CAS  Google Scholar 

  • Wright AG (2006) Phylogenetic relationships within the order Halobacteriales inferred from 16S rRNA gene sequences. Int J Syst Evol Microbiol 56:1223–1227

    Article  Google Scholar 

  • 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(1):10

    Article  CAS  Google Scholar 

  • Zhang WJ, Cui HL (2014) Halorubrum salinum sp. nov., isolated from a marine solar saltern. Arch Microbiol 196(6):395–400

    Article  CAS  Google Scholar 

  • Zhang Y, Hao J, Zhang YQ, Chen XL, Xie BB, Shi M, Li PY (2017) Identification and characterization of a novel salt-tolerant esterase from the deep-sea sediment of the South China Sea. Front Microbiol 8:441

    Google Scholar 

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Acknowledgements

This work was supported by BITS Pilani Seed Grant 2013 to JB. DD would like to thank BITS Pilani, KK Birla Goa Campus for the fellowship. The authors thank Dr Hiroaki Minegishi, Bio-Nano Electronics Research Center, Tokyo University, Japan for identifying the haloarchaeal isolates.

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Correspondence to Judith M. Braganca.

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Das, D., Kalra, I., Mani, K. et al. Characterization of extremely halophilic archaeal isolates from Indian salt pans and their screening for production of hydrolytic enzymes. Environmental Sustainability 2, 227–239 (2019). https://doi.org/10.1007/s42398-019-00077-x

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