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Accumulation of lipid in Dunaliella salina under Nutrient Starvation Condition

Truc Mai , Phuc Nguyen, Trung Vo , Hieu Huynh, Son Tran, Tran Nim, Dat Tran, Hung Nguyen, Phung Bui
American Journal of Food and Nutrition. 2017, 5(2), 58-61. DOI: 10.12691/ajfn-5-2-2
Published online: April 22, 2017

Abstract

The effect of nutrient starvation on lipid accumulation of Dunaliella salina A9 was studied. In nutrient starvation, cell colour changed from green to yellow (or orange) and cell growth reached stationary phase after 9 days of the culture. The study showed that under nutrient stress, decreased in cell growth is accompanied by carotenoid biosynthesis and lipid content of Dunaliella salina. The results of this study can be used to increase carotenoid and lipid production in microalgae for functional food and biofuel in the future.

1. Introduction

Dunaliella currently belongs to the order of Chlamydomonadales, family Dunaliellaceae, according to NCBI database 28. Almost 200 years from its discovery in 1838 by Michel Felix Dunal, the genus has received great taxonomic treatment with 22 marine and halophilic species found and described. The genus is well characterized in term of habitat and cell cycle 6. Later, D. salina is the most well-studied species in this genus, enjoying great attention as it has been considered an economically-efficient method to produce β-carotene, capable of accumulating more than 10% of cells dry biomass under optimal stress-inducing conditions 7. Other than β-carotene, the species can accumulate good amount of secondary metabolites in industrial scale culture 20. These secondary metabolites are produced as the organism response to different stress condition, for example glycerol is produced after salt-stress induction 12, 20, 24, neutral lipids and antioxidants (e.g. β-carotene) in response to macronutrient starvation 9, 21, 22, 41 and high light intensity 2, 13, 18; production of carotenoids, especially α-carotene and 9-cis β-carotene 10, 17, 25 in low temperature, and can intensify in the presence of multiple stresses 1, 2.

Of different stressors, macronutrient limitation appears to be the main regulatory factor controlling neutral lipid accumulation 33, 35, 40. In nutrient depletion condition, D. salina accumulates intra- or extra-plastidic lipid bodies composed of both TAGs (triacylglycerides) and carotenoids 2, 14. Previous finding has indicated that β-carotene synthesis and lipid deposition is inter-dependent; biosynthesis of β-carotene is suppressed when lipid metabolism pathway is inhibited 30. Bonnefond et al. 5 found that β-carotene, in particular, has a positive relationship with TAGs synthesis when N:C ratio in medium decreases. Currently, TAGs are targets for biodiesel production 11, 16, 19, 34. Production of biofuel from algae is dependent on microalgal biomass production rate and lipid content. Both biomass production and lipid accumulation are limited by several factors, of which nutrients play a key role. In earlier studies, we were interested in screening Vietnam’s marine algae flora in search for a potential producer of β-carotene, TAGs and other valuable compounds 37, 38, 39 and compare their production to D. bardawil, a global source for bioactive compounds production 37, 39. We have found that a local strain named D.salina A9 has considerable concentration of β-carotene per volume culture and higher growth rate compared to D. bardawil 39, which may confer its competitive lipid producer. This study is a continuation of previous effort to examine the strain’s efficacy in lipid production.

2. Materials and Methods

2.1. Dunaliella salina Strains and Experiments

The experiments were carried out on 2 Dunaliella salina strains including Dunaliella salina A9 isolated at Department of Algal Biotechnology, International University, Viet Nam and Dunaliella salina var. bardawil DCCBC 15 (D. bardawil) kindly provided by Dr. E.W. Polle, Department of Biology, Brooklyn College of CUNY Brooklyn, NY (USA).

The algae were grown in the MD4 medium (1.5M NaCl) according to Tran et al. 36. Briefly, the medium contained natural seawater, and was added with NPK 0.1 g/L, MgSO4 1.86 g/L, EDTA 0.00876 g/L, FeCl3 0.00049 g/L, MnCl2 0.00189 g/L, NaHCO3 50mM, pH = 7.5. Dunaliella strains were cultivated at light intensity 50 μmol photon/m2/sec and continuous light in 50 ml flasks at 25°C temperature during 15 days. Each strain was triplicate in each experiment, and all experiments were repeated at least twice.

2.2. Growth Analysis

Lugol solution (5% iodine and 10% potassium iodide mixed in distilled water) was used to stop algae movement. Cell density was estimated by cell count every three days, using a light microscope with 0.1 mm deep counting chamber (Neubauer Haemocytometer). Cell number was determined by following formula: Number of cells/ml = total cells counted x 104 x dilution factor.

2.3. Sulfo-phospho-vanillin Assay for Lipid Estimation

Phosphovanillin reagent was prepared by initially dissolving 0.06 g vanillin in 2 mL absolute ethanol; 8 mL deionized water and stirred continuously. Subsequently 50 ml of concentrated H2SO4 was added to the mixture, and the resulting reagent was stored in the dark until use. To ensure high activity, fresh phospho-vanillin reagent was prepared shortly before every experiment 23.

For SPV reaction of the algal culture for lipid quantification, 1 mL of algal suspension was centrifuged at 5000 rpm for 5 min and the pellet was extracted with 2 mL of concentrated (98%) H2SO4. The mixture was then heated for 10 min at 100°C, and cooled for 5 min in ice bath. 5 mL of freshly prepared phospho-vanillin reagent was then added, and the sample was incubated for 15 min at 37°C incubator shaker at 200 rpm. Absorbance reading at 530 nm was taken in order to quantify lipid content of the sample 23.

2.4. Data Analysis

Data was processed in Excel and analyzed by one-way ANOVA using SPSS software. All significant levels were set at p < 0.05.

3. Results and discussion

3.1. Cell Morphology

Dunaliella salina cells were green in exponential growth phase from day 0 to day 9, however they turned yellow or orange and cell size increased significantly in stationary growth phase (after 9 days of culture (Figure 1). Microscopy showed carotenoid accumulation of D. salina increased under condition of nutrient starvation in the stationary phase. Dunaliella cells may change shape with changing conditions, often becoming spherical under unfavourable conditions. Cell size may vary to some degree with growth conditions and light intensity. Chloroplast can accumulate large quantities of β-carotene within oily globules in the inter-thylakoid spaces, thus cell becoming orange-red rather than green. The β-carotene globules of Dunaliella salina were found to be composed of practically only neutral lipids, more than half of which were β-carotene 2.

3.2. The Growth and Lipid Accumulation of Dunaliella salina

Dunaliella salina A9 reached stationary phase of growth after 9 days (Figure 2). Lipid accumulation (lipid per ml and lipid per cell) increased during culture. In particular, significantly higher lipid content was obtained after 12 days of growth (Figure 2), corresponding to the observation of carotene-rich globules (Figure 1). For Dunaliella bardawil, maximum cell number obtained at day 6 and then decreased (Figure 3). However, lipid accumulation increased significantly after 9 days (Figure 3). The nutrient starvation in the stationary phase led to lipid accumulation and carotenoid in chloroplast of cells earlier in D. bardawil than in D.salina A9.

The lipid content in microalgae varies from 1-85% of dry weight and is effect by the nutritional composition of the medium. Lipid accumulation in algae typically occurs during periods of environmental stress as the nutrient deficient condition. Dunaliella species respond to nitrogen starvation by increasing lipid production 8.

Vanitha et al. 2007 compared lipid profiles between the D.salina and D. bardawil and found that they are very different. While neutral lipids do not differ significantly between the two, polar lipids in D.bardawil is significantly lower than D. salina, comprising of 24.2 % compared to 42.52% of total lipids in D. salina, while glycerolipids content in D.bardawil is much higher (40.42% total lipid compared to 28.4 % in D. salina). This may suggest that the two species behave differently under the same stress condition, shifting their lipid composition to serve alternative counter-stress mechanisms. Comparing lipid/cell and lipid/mL of the two cultures, are they significantly different?

In this study, we found that increase in β-carotene synthesis in D.salina A9 under stress condition corresponds to increase in total lipid concentration, which agrees with previous studies 5, 30, 37. Other research has found that TAGs abundance over total lipid increases with Nitrogen depletion and β-carotene synthesis, while polar lipids composition decreases 5. This might be explained by new findings about new roles of TAGs besides storage organelles, and as constitutors of intermediary products in biosynthesis of more complex molecules such as glycolipids and phospholipids 15. In the meantime, abundance of polar lipids correspond to the minimal availability for cell and organelle membranes 5.

4. Conclusion

D. salina can accumulate higher carotenoid and lipid contents under stress conditions, such as nutrient starvation, salinity and light. Lipid from microalgae is supplied in food, feed and biofuel production. Dunaliella species have been used as global source of lipids production for biodiesel and carotenoids 3. Previously, a strain of D.salina collected from salt pan in Bombay, India was reported to have prominent concentration of eicosapentanoeic acid (EPA) 4 as high as 21.4%. EPA is part of a series of bioactive polyunsaturated fatty acids (PUFAs). Commonly known PUFAs such as eicosapenaeoic (EPA), docosahexanenoic (DHA), arachidonic acid (AA), α-linoleic (ALA) and γ-linoleic (GLA), also loosely referred to as omega-3 and omega-6 fatty acids, have anti-inflammatory and inflammatory related diseases, as potent vasodilators or anti-coagulation properties 29, hypertension, diabetes, coronary heart disease, skin diseas, etc. 26, 27, 31, 32. As a result of this study, we were able to demonstrate that Dunaliella salina A9 increased lipid production under nutrient starvation condition. Our future direction would be to explore the lipid content of this strain under different stress conditions for novel bioactive compounds and their maximal capacity.

References

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In article      View Article
 
[2]  Ben-Amotz, A. and Avron, M., “On the factors which determine massive β-carotene accumulation in the halotolerant alga Dunaliella bardawil”, Plant Physiol., 1983, 72. 593-597.
In article      View Article  PubMed
 
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In article      View Article
 
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In article      View Article
 
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In article      View Article
 
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In article      View Article  PubMed
 
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In article      View Article
 
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In article      View Article
 
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In article      View Article  PubMed
 
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In article      View Article
 
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In article      View Article  PubMed
 
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In article      View Article
 
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In article      View Article  PubMed
 
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Truc Mai, Phuc Nguyen, Trung Vo, Hieu Huynh, Son Tran, Tran Nim, Dat Tran, Hung Nguyen, Phung Bui. Accumulation of lipid in Dunaliella salina under Nutrient Starvation Condition. American Journal of Food and Nutrition. Vol. 5, No. 2, 2017, pp 58-61. http://pubs.sciepub.com/ajfn/5/2/2
MLA Style
Mai, Truc, et al. "Accumulation of lipid in Dunaliella salina under Nutrient Starvation Condition." American Journal of Food and Nutrition 5.2 (2017): 58-61.
APA Style
Mai, T. , Nguyen, P. , Vo, T. , Huynh, H. , Tran, S. , Nim, T. , Tran, D. , Nguyen, H. , & Bui, P. (2017). Accumulation of lipid in Dunaliella salina under Nutrient Starvation Condition. American Journal of Food and Nutrition, 5(2), 58-61.
Chicago Style
Mai, Truc, Phuc Nguyen, Trung Vo, Hieu Huynh, Son Tran, Tran Nim, Dat Tran, Hung Nguyen, and Phung Bui. "Accumulation of lipid in Dunaliella salina under Nutrient Starvation Condition." American Journal of Food and Nutrition 5, no. 2 (2017): 58-61.
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[1]  Ben-Amotz, A., “Effect of irradiance and nutrition deficiency on the chemical composition of D. bardawil (Volvocales, Chlorophyta)”, J. Plant Physiol., 1987), 131. 479-487.
In article      View Article
 
[2]  Ben-Amotz, A. and Avron, M., “On the factors which determine massive β-carotene accumulation in the halotolerant alga Dunaliella bardawil”, Plant Physiol., 1983, 72. 593-597.
In article      View Article  PubMed
 
[3]  Ben-Amotz, A., Katz, A. and Avron, M., “Accumulation of ß-carotene in halotolerant algae: purification and characterization of β-carotene-rich globules from Dunaliella bardawil (Chlorophyceae)” J. Phycol., 1982, 18. 529-537.
In article      View Article
 
[4]  Bhosale, R. A., Rajabhoj, M. P. and Chaugule, B. B., “Dunaliella salina Teod. as a prominent source of Eicosapentaenoic acid”, Inter. J. Algae, 2010, 12 (2). 185-189.
In article      View Article
 
[5]  Bonnefond, H., Moelants, N., Talec, A., Mayzaud, P., Bernard, O. and Sciandra, A., “Coupling and uncoupling of triglyceride and β-carotene production by Dunaliella salina under nitrogen limitation and starvation”, Biotechnol Biofuels, 2017, 10 (25).
In article      View Article
 
[6]  Borowitzka, M. and Siva, J., “The taxonomy of the genus Dunaliella (Chlorophyta, Dunaliellales) with emphasis on the marine and halophilic species”, Appl Phycol., 2007, 19(5). 567-590.
In article      View Article
 
[7]  Borowitzka, M., “Microalgae as sources of pharmaceuticals and other biologically active compounds”, J Appl. Phycol., 1995, 7. 3-15.
In article      View Article
 
[8]  Chen, M., Tang, H., Ma, H., Holland, T. C., Ng, K.Y. S., Salley, S. O., “Effect of nutrients on growth and lipid accumulation in the green algae Dunaliella tertiolecta”, Bioresource Technology, 2011, 102. 1649-1655.
In article      View Article  PubMed
 
[9]  Gao, Y., Yang, M., Wang, C., “Nutrient deprivation enhances lipid content in marine microalgae”, Bioresource Technology, 2013, 147. 484-491.
In article      View Article  PubMed
 
[10]  Gómez, P. I. and González, M. A., “The effect of temperature and irradiance on the growth and carotenogenic capacity of seven strains of Dunaliella salina (Chlorophyta) cultivated under laboratory condition”, Biol. Res., 2005, 38(2-3). 151-162.
In article      View Article  PubMed
 
[11]  Griffiths, M. and Harrison, S., “Lipid productivity as a key characteristic for choosing algal species for biodiesel production”, J. Appl. Phycol., 2009, 21. 493-507.
In article      View Article
 
[12]  Hadi, M. R., Shariati, M., Afsharzadeh, S., “Microalgal biotechnology: carotenoid and glycerol production by the green algae Dunaliella isolated from the Gave-Khooni salt marsh, Iran”, Biotechnol. Bioproc. Eng., 2008, 13. 540-544.
In article      View Article
 
[13]  Hejazi, M. A. and Wijffels, R. H., “Effect of light intensity on β-carotene production and extraction by Dunaliella salina in two-phase bioreactors”, Biomolecular Engineering, 2003, 20(4-6). 171-175.
In article      View Article
 
[14]  Jiménez, C. and Pick, U., “Differential stereoisomer composition of β, β-carotene in thylakoids and in pigment globules in Dunaliella”, J. Plant Physiol., 1994, 143. 257-263.
In article      View Article
 
[15]  Khozin-Goldberg, I., Yu, H. Z., Adlerstein, D., Didi-Cohen, S., Heimer, Y. M., Cohen, Z., “Triacylglycerols of the red microalga Porphyridium cruentum can contribute to the biosynthesis of eukaryotic galactolipids”, Lipids, 2000, 35. 881-889.
In article      View Article  PubMed
 
[16]  Kim, J., Yoo, G., Lee, H., Lim, J., Kim, K., Kim, C. W. and Park, M. S., “Methods of downstream processing for the production of biodiesel from microalgae”, Biotechnology Advances, 2013, 31 (6). 862-876.
In article      View Article  PubMed
 
[17]  Krót, M., Maxwell, D. P., Huner, P. A., “Exposure of Dunaliella salina to low temperature mimics the high light-induced accumulation of Carotenoids and the carotenoid binding proteins (Cbr)”, Plant Cell Physiol., 1997, 38(2). 213-216.
In article      View Article
 
[18]  Lamers, P. P., van de Laak, C. C., Kaasenbrood, P. S., Lorier, J., Janssen, M., De Vos, R. C., Bino, R. J., Wijffels, R. H., “Carotenoid and fatty acid metabolism in light-stressed Dunaliella salina”, Biotechnology and Bioengineering, 2010, 106(4). 638-48.
In article      View Article  PubMed
 
[19]  Li, Y., Horsman, M., Wu, N., Lan, C.Q. and Dubois-Calero, N., “Biofuels from microalgae”, Biotechnol.Prog., 2008, 24. 815-820.
In article      View Article
 
[20]  Lemoine, Y. and Schoefs, B., “Secondary ketocarotenoid astaxanthin biosynthesis in algae: a multifunctional response to stress”, PsynR, 2010, 106. 155-157.
In article      View Article
 
[21]  Lv, H., Cui, X., Wahid, F., Xia, F., Zhong, C., Jia, S., “Analysis of the physiological and molecular responses of Dunaliella salina to macronutrient deprivation”, PLOS ONE, 2016, 11(3). 1-19.
In article      View Article  PubMed
 
[22]  Mendoza, H., Martel, A., Jiménez del Río, M. and García Reina, G., “Oleic acid is the main fatty acid related with carotenogenesis in Dunaliella salina” J. Appl. Phycol., 1999, 11. 15-19.
In article      View Article
 
[23]  Mishra S. K., Suh, W. I., Farooq, W., Moon, M., Shrivastav, A., Park, M. S., Yang, J. W., “Rapid quantification of microalgal lipids in aqueous medium by a simple colorimetric method”, Bioresource Technology, 2014, 155. 330-333.
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