Physiological characterization and stress-induced metabolic responses of Dunaliella salina isolated from salt pan


A Dunaliella strain was isolated from salt crystals obtained from experimental salt farm of the institute (latitude 21.46 N, longitude 72.11°E). The comparative homology study of amplified molecular signature 18S rRNA, proves the isolated strain as D. salina. The growth pattern and metabolic responses such as proline, glycine betaine, glycerol, total protein and total sugar content to different salinity (from 0.5 to 5.5 M NaCl) were studied. The optimum growth was observed at 1.0 M NaCl and thereafter it started to decline. Maximum growth was obtained on 17th day of inoculation in all salt concentrations except 0.5 M NaCl, whereas maximum growth was observed on 13th day. There were no significant differences (P < 0.01) in chlorophyll a/b contents (1.0–1.16 ± 0.05 μg chl. a and 0.2–0.29 ± 0.01 μg chl. b per 106 cells) up to 2.0 M NaCl, however at 3.0 M NaCl a significant increase (2.5 ± 0.12 μg chl. a and 0.84 ± 0.4 μg chl. b per 106 cells) was observed which declined again at 5.5 M NaCl concentration (2.0 ± 0.1 μg chl. a and 0.52 ± 0.03 μg chl. b per 106 cells). Stress metabolites such as proline, glycine betaine, glycerol and total sugar content increased concomitantly with salt concentration. Maximum increase in proline (1.4 ± 0.07 μg), glycine betaine (5.7 ± 0.28 μg), glycerol (3.7 ± 0.18 ml) and total sugar (250 ± 12.5 μg) per 105 cells was observed in 5.5 M NaCl. A decrease in total protein with reference to 0.5 M NaCl was observed up to 3.0 M NaCl, however, a significant increase (P < 0.01) was observed at 5.5 M NaCl (0.19 ± 0.01 μg per 105 cells). Inductive coupled plasma (ICP) analysis shows that intracellular Na+ remained unchanged up to 2.0 M NaCl concentration and thereafter a significant increase was observed. No relevant increase in the intracellular level of K+ and Mg++ was observed with increasing salt concentration. Evaluation of physiological and metabolic attributes of Dunaliella salina can be used to explore its biotechnological and industrial potential.

This is a preview of subscription content, log in to check access.

Access options

Buy single article

Instant unlimited access to the full article PDF.

US$ 39.95

Price includes VAT for USA

Subscribe to journal

Immediate online access to all issues from 2019. Subscription will auto renew annually.

US$ 199

This is the net price. Taxes to be calculated in checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5


  1. 1.

    Agong SG, Yoshida Y, Yazawa S, Masuda M (2004) Tomato response to salt stress. Acta Hort (ISHS) 637:93–97

  2. 2.

    Amotz AB, Avron M (1973) The role of glycerol in the osmotic regulation of the halophilic alga Dunaliella parva. Plant Physiol 51:875–878

  3. 3.

    Archbald HK (1940) Fructosans in the monocotyledons, a review. New Phytol 39:185–219

  4. 4.

    Avron A (1986) The osmotic component of halotolerant algae. Trends Biochem Sci 11:5–6

  5. 5.

    Bates LS, Waldren RP, Teare ID (1973) Rapid determination of free proline for water stress studies. Plant Soil 39:205–207

  6. 6.

    Ben-Amotz A, Avron M (1990) The biotechnology of cultivating the halotolerant algae Dunaliella. Tibtech 8:121–125

  7. 7.

    Bohnert HJ, Nelson DE, Jonsen RG (1995) Adaptations to environmental stresses. Plant Cell 7:1099–1111

  8. 8.

    Borowitzka MA, Siva CJ (2007) The taxonomy of the genus Dunaliella (Chlorophyta, Dunaliellales) with emphasis on the marine and halophilic species. J Appl Phycol 19:567–590

  9. 9.

    Chitlaru E, Pick U (1991) Regulation of glycerol synthesis in response to osmatic changes in Dunaliella. Plant Physiol 96:50–60

  10. 10.

    Cifuentes AS, González MA, Inostroza I, Aguilera A (2001) Reappraisal of physiological attributes of nine strains of Dunaliella (chlorophyceae): growth and pigment content across a salinity gradient. J Phycol 37:334–344

  11. 11.

    Cowan AK, Rose PD, Horne LG (1992) Dunaliella salina: a model system for studying the response of plant cells to stress. J Exp Bot 43:1535–1547

  12. 12.

    Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA minipreparation: version II. Plant Mol Biol Rep 1:19–21

  13. 13.

    Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F (1956) Colorimetric method for determination of sugars and related substances. Anal Chem 28:350–356

  14. 14.

    Ehrenfeld J, Cousin JL (1982) Ionic regulation of the unicellular green alga Dunaliella tertiolecta. J Membr Biol 70:47–57

  15. 15.

    Ehrenfeld J, Cousin JL (1984) Ionic regulation of the unicellular green alga Dunaliella tertiolecta: response to hypertonic shock. J Membr Biol 77:45–55

  16. 16.

    Elevi BR, Khristo P, Oren A (2008) Interrelationships between Dunaliella and halophilic prokaryotes in saltern crystallizer ponds. Extremophiles 12:5–14

  17. 17.

    Epstein E, Rush JD, Kingsbury RW, Kelley DB, Cinnigham GA, Wrono AF (1980) Saline culture of crops: a genetic approach. Science 210:399–404

  18. 18.

    Fazeli MR, Tofighi H, Samadi N, Jamalifar H (2006) Effects of salinity on β-carotene production by Dunaliella tertiolecta DCCBC26 isolated from the Urmia salt lake, north of Iran. Bioresour Technol 97:2453–2456

  19. 19.

    Ferris MJ, Hirsch CF (1991) Method for isolation and purification of cyanobacteria. Appl Environ Microbiol 57:1448–1452

  20. 20.

    Garcίa F, Freile-Pelegrίn Y, Robledo D (2007) Physiological characterization of Dunaliella sp. (Chlorophyta, Volvocales) from Yucatan, Mexico. Bioresour Technol 98:1359–1365

  21. 21.

    Geng D, Wang Y, Wang P, Li W, Sun Y (2003) Stable expression of hepatitis B surface antigen gene in Dunaliella salina (Chlorophyta). J Appl Phycol 15:451–456

  22. 22.

    Gill PK, Sharma AD, Singh P, Bhullar SS (2001) Effect of various abiotic stresses on the growth, soluble sugars and water relations of sorghum seedlings grown in light and darkness. Bulg J Plant Physiol 27:72–84

  23. 23.

    Gimmler H, Schirling R (1978) Cation permeability of the plasmalemma of the halotolerant alga Dunaliella parva. II. Cation content and glycerol concentration of the cells as dependent upon external NaCl concentration. Z Pflanzenphysiol 87:435–444

  24. 24.

    Ginzburg M (1981) Measurements of ion concentrations in Dunaliella parva subjected to hypertonic shocks. Y Exp Bot 32:333–340

  25. 25.

    Gómez PI, González MA (2005) The effect of temperature and irradiance on the growth and carotenogenic capacity of seven strains of Dunaliella salina (Chlorophyta) cultivated under laboratory conditions. Biol Res 38:151–162

  26. 26.

    Gónzalez MA, Coleman AW, Gómez PI, Montoya R (2001) Phylogenetic relationship among various strains of Dunaliella (Chlorophyceae) based on nuclear ITS rDNA sequences. J Phycol 37:604–611

  27. 27.

    Grieve CM, Grattan SR (1983) Rapid assay for determination of water soluble quaternary ammonium compounds. Plant Soil 70:303–307

  28. 28.

    Guillard RRL (2005) Purification methods for microalgae. In: Andersen RA (ed) Algal culturing techniques. Elsevier Academic Press, San Diego, pp 117–132

  29. 29.

    Hare PD, Cress WA, Van-Staden J (1998) Dissecting the roles of osmolyte accumulation during stress. Plant Cell Environ 21:535–554

  30. 30.

    Hasegawa PM, Bressan RA, Zhu JK, Bohnert HJ (2000) Plant cellular and molecular responses to high salinity. Annu Rev Plant Physiol Plant Mol Biol 51:463–499

  31. 31.

    Hong B, Barg R, Ho TH (1992) Developmental and organ specific expression of an ABA- and stress-induced protein in barley. Plant Mol Biol 18:663–674

  32. 32.

    Hoque MA, Okuma E, Banu MNA, Nakamura Y, Shimoishi Y, Murata Y (2007) Exogenous proline mitigates the detrimental effects of salt stress more than exogenous betaine by increasing antioxidant enzyme activities. J Plant Physiol 164:553–561

  33. 33.

    Jeffrey SW, Humphrey GF (1975) New spectrophotometric equations for determining chlorophyll a, b, c1 and c2 in higher plants and natural phytoplankton. Bioch Physiol Pflanz (BPP) 165:191–194

  34. 34.

    Kaplan A, Schreiber U, Avron M (1980) Salt-induced metabolic changes in Dunaliella salina. Plant Physiol 65:810–813

  35. 35.

    Katz A, Avron M (1985) Determination of intracellular osmotic volume and sodium concentration in Dunaliella. Plant Physiol 78:817–820

  36. 36.

    Katz A, Bental M, Degani H, Avron M (1991) In vivo pH regulation by a Na+/H+ antiporter in the halotolerant alga Dunaliella salina. Plant Physiol 96:110–115

  37. 37.

    Kawasaki S, Borchert C, Deyholos M, Wang H, Brazille S, Kawai K, Galbraith D, Bohnert HJ (2001) Gene expression profiles during the initial phase of salt stress in rice. Plant Cell 13:889–905

  38. 38.

    Khedr AHA, Abbas MA, Wahid AAA, Quick WP, Abogadallah GM (2003) Proline induces the expression of salt-stress-responsive proteins and may improve the adaptation of Pancratium maritimum L. to salt-stress. J Exp Bot 54:2553–2562

  39. 39.

    Lambert M, Neish AC (1950) Rapid method for estimation of glycerol in fermentation solutions. Can J Res 28:83–89

  40. 40.

    Li Q, Gao X, Sun Y, Zhang Q, Sang R, Xu Z (2006) Isolation and characterization of a sodium- dependent phosphate transporter gene in Dunaliella viridis. Biochem Biophys Res Commun 340:95–104

  41. 41.

    Liska AJ, Shevchenko A, Pick U, Katz A (2004) Enhanced photosynthesis and redox energy production contribute to salinity tolerance in Dunaliella as revealed by homology-based proteomics. Plant Physiol 136:2806–2817

  42. 42.

    Lowery OH, Rosenbrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent. J Biol Chem 193:265–275

  43. 43.

    Meier H, Reid JSD (1982) Reserve polysaccharides other than starch in higher plants. In: Loewus FA, Tanner W (eds) Encyclopedia of plant physiology, new series, vol 13a. Springer-Verlag, Berlin, pp 418–471

  44. 44.

    Muñoz J, Mudge SM, Sandoval A (2004) Effects of ionic strength on the production of short chain volatile hydrocarbons by Dunaliella salina (Teodoresco). Chemosphere 54:1267–1271

  45. 45.

    Olmos J, Paniagua J, Contreras R (2000) Molecular identification of Dunaliella sp. utilizing the 18S rDNA gene. Lett Appl Microbiol 30:80–84

  46. 46.

    Oren A (2005) A hundred years of Dunaliella research: 1905–2005. Saline Syst 1:2

  47. 47.

    Orset S, Young AJ (1999) Low-temperature-induced synthesis of α-carotene in the microalga Dunaliella salina (chlorophyta). J Phycol 35:520–527

  48. 48.

    Ozturk ZN, Talame V, Deyholos M, Michalowski CB, Galbraith DW, Gozukirmizi N, Tuberosa R, Bohnert HJ (2002) Monitoring large-scale changes in transcript abundance in drought- and salt-stressed barley. Plant Mol Biol 48:551–573

  49. 49.

    Phadwal K, Singh PK (2003) Effect of nutrient depletion on β-carotene and glycerol accumulation in two strains of Dunaliella sp. Bioresour Technol 90:55–58

  50. 50.

    Phadwal K, Singh PK (2003) Isolation and characterization of an indigenous isolate of Dunaliella sp. for β-carotene and glycerol production from a hypersaline lake in India. J Basic Microbiol 43:423–429

  51. 51.

    Pick U (1992) ATPases and ion transport in Dunaliella. In: Avron M, Ben-Amtoz A (eds) Dunaliella: physiology, biochemical and biotechnology. CRC press, Boca Raton, pp 63–97

  52. 52.

    Prado FE, Boero C, Gallardo M, Gonzalez JA (2000) Effect of NaCl on germination, growth and soluble sugar content in Chenopodium quinoa wild seeds. Bot Bull Acad Sin 41:27–34

  53. 53.

    Raja R, Iswarya SH, Balasubramanyam D, Rengasamy R (2007) PCR- identification of Dunaliella salina (Volvocales, Chlorophyta) and its growth characteristics. Microbiol Res 162:168–176

  54. 54.

    Riisgard HU, Norgard-Nielsen K, Sogaard-Jensen B (1980) Further studies on volume regulation and effects of copper in relation to pH and EDTA in the naked marine flagellate Dunaliella marina. Mar Biol 56:267–276

  55. 55.

    Seki M, Ishida J, Narusaka M, Fujita M, Nanjo T, Umezawa T, Kamiya A, Nakajima M, Enju A, Sakurai T (2002) Monitoring the expression pattern of around 7, 000 Arabidopsis genes under ABA treatments using a full-length cDNA microarray. Funct Integr Genomics 2:282–291

  56. 56.

    Shaish A, Ben-Amotz A, Avron M (1991) Production and selection of high β-carotene mutants of D. bardawil (chlorophyta). J Phycol 27:652–656

  57. 57.

    Sokal RR, Rohlf FJ (1995) Biometry, the principles and practice of statistics in biological research, 3rd edn edn. WH Freeman and Company, New York, pp 321–356

  58. 58.

    Szekely G (2004) The role of proline in Arabidopsis thaliana osmotic stress response. Acta Biol Szeged 48:81

  59. 59.

    Takagi M, Karseno Yoshida T (2006) Effect of salt concentration on intracellular accumulation of lipids and triacylglyceride in marine microalgae Dunaliella cells. J Biosci Bioeng 101:223–226

  60. 60.

    Thakur A, Kumar HD, Cowsik SM (2000) Effect of pH and inorganic carbon concentration on growth, glycerol production, photosynthesis and dark respiration of Dunaliella salina. Cytobios 102:69–74

  61. 61.

    Vaara T, Vaara M, Niemela S (1979) Two improved methods for obtaining axenic cultures of cyanobacteria. Appl Environ Microbiol 38:1011–1014

  62. 62.

    Weisburg WG, Barns SM, Pelletier DA, Lane DJ (1991) 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173:697–703

  63. 63.

    Wilcox LW, Lewis A, Fuerst PA, Floyd GL (1992) Group I introns within the nuclear-encoded smallsubunit rRNA gene of three green algae. Mol Biol Evol 9:1103–1118

  64. 64.

    Yancey PH (1994) Compatible and counteracting solutes. In: Strange K, Boka R (eds) Cellular and molecular physiology of cell volume regulation. CRC press, Boca Raton, pp 82–109

  65. 65.

    Yancey PH, Clark ME, Hand SC, Bowlis RD, Somero GN (1982) Living with water stress: evolution of osmolyte system. Science 217:1214–1222

Download references


The authors are grateful to Director, Central Salt and Marine Chemicals Research Institute, Bhavnagar, Gujarat for his support, encouragement and providing research facilities. The financial support of CSIR, Govt. of India, GSBTM, Govt. of Gujarat and Department of Science and Technology, Govt. of India (Under SERC Fast Track scheme vide Order No.: SR/FT/L-25/2005 dated 2 January 2006) for carrying out this project is thankfully acknowledged.

Author information

Correspondence to Avinash Mishra.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 2 (WMV 1229 kb)

Supplementary material 3 (WMV 4760 kb)

Supplementary material 1 (JPG 42.4 kb)

Supplementary material 2 (WMV 1229 kb)

Supplementary material 3 (WMV 4760 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Mishra, A., Mandoli, A. & Jha, B. Physiological characterization and stress-induced metabolic responses of Dunaliella salina isolated from salt pan. J Ind Microbiol Biotechnol 35, 1093 (2008) doi:10.1007/s10295-008-0387-9

Download citation


  • Dunaliella
  • Proline
  • Glycine betaine
  • Glycerol
  • Metabolites
  • Salinity