Int J Pharm Pharm Sci, Vol 9, Issue 4, 32-37Original Article


COLOMBIAN STRAIN OF DUNALIELLA SALINA AS A SOURCE OF METABOLITES OF HIGH COMMERCIAL VALUES

GALLEGO-CARTAGENA EULERa, CASTILLO-RAMÍREZ MARGARITAa, MARTÍNEZ-BURGOS WALTERb

aResearch Group Environmental Management and Sustainability GESSA, Faculty of Environmental Sciences, Universidad de La Costa, Barranquilla 3599481, Atlántico, Colombia, bFaculty of Enginering, Universidad del Norte, Barranquilla 3509509, Atlántico, Colombia
Email: egallego1@cuc.edu.co

Received: 27 Oct 2016 Revised and Accepted: 14 Feb 2017


ABSTRACT

Objective: The objective was to evaluate the effect of stressful and non-stressful conditions on the growth and production of pigments in a strain of Dunaliella salina (D. salina) isolated from the artificial saline of Manaure municipality, department of La Guajira, Colombia in laboratory conditions.

Methods: Two treatments were performed, one in non-stressful growing conditions with medium J/1 to 1 M NaCl, 190-µmol. m-2. s-1, 5.0 mmol KNO3, pH 8.2 and another in stressful conditions with medium J/1 to 4.0 M NaCl, 390 µmol. m-2. s-1, 0.50 mmol KNO3, each in triplicate. Population growth was assessed by cell count, and the pigment content was performed by spectrophotometric techniques.

Results: It was found that the conditions of stressful influences in the growth and the production of carotenoids of D. salina in comparison with those cultures not stressed. There was a significant difference between the average values of total carotenoids in the experiment with stressful conditions with 9.67±0.19 µg/ml and the experiment with conditions not stressful with 1.54±0.08 µg/ml at the level of significance of p<0.05.

Conclusion: It was demonstrated that the stressful condition in the culture is associated with an increase in the production of lipophilic antioxidants, among these carotenoids. The knowledge of the stressful conditions for the production of carotenoids from D. salina isolated from the saline of Manaure opens a field in the use of this biotic resource with biotechnological purposes, production of new antibiotics, nutraceuticals and/or production of biofuels.

Keywords: Dunaliella salina, Carotenoides, Saline of Manaure, Stressful conditions, Biotechnological purposes


INTRODUCTION

The prospecting of microalgae in the course of the last four decades has been diversifying due to their industrial applications, especially as raw material to obtain bioproduct chemicals with pharmaceutical purposes, therapeutic [1], cosmetic, nutraceutical, animal food concentrates [2], for the domestic waste-water treatment, bio fertilizers [3] human food [4], live food for aquaculture organisms [5], and obtaining biofuels [6, 7]. The advantage of using microalgae lies mainly in its photosynthetic capacity and metabolic plasticity, which has served as the basis for ambitious challenges and implementation of technological strategies for its cultivation. The advances in techniques and procedures of genetic engineering and biology have allowed the selection and improvement of strains adapted to stressful conditions of cultivation [8, 9]. This has been for the industries of bioenergy, bio products and healthy food, a decrease in costs according to the competitiveness of the new markets [10]. Most of the biomass produced in the field of industrial microalgae biotechnology, is around 107 tons per year [11], and is mainly obtained from species of genus Botryococcus, Chlorella, Dunaliella, Haematococcus and Spirulina, which are cultivated for the production of proteins, astaxanthin, ß-carotene, glycerol, liquid fuels and pharmaceutical formulations and even products of fine chemistry [12-14]. Although most of the research has focused on the production of carotenoids from microalgae due to this type of compound in terms of real or potential industrial applications, this represents an interest for the production of food, cosmetics and vitamins with antioxidant properties.

In effect, this has generated a strong demand in the market where the price of β-carotene of microalgae reaches a commercial value of 700 €/kg, while its counterpart synthetic does not reach half of that [15]. The preference of β-carotene natural -a secondary metabolite- that consists of a mixture of stereoisomers 9 cis and trans, with 40 and 50% respectively for the health market is due to its properties as a stimulator of the immune system and its uses in the treatment of more than 60 deadly diseases, including various forms of cancer, heart disease and neurodegenerative diseases, premature aging and arthritis [16, 17] and pathologies with hyperlipidemia and hyper-cholesterolemia effects [18, 19].

The Dunaliella genus is represented by two species, D. salina and D. viridis, which are unicellular eukaryotic organisms with naked cells, flagella isokont and a pirenoide; present as a chloroplast with cup shape; contain chlorophyll a and b, xanthophylls (violaxanthin, zeaxanthin and lutein, etc.), carotenes (β, α and γ), sometimes with structures of resistance or aplanospores; the regular reproduction is by longitudinal division, or are also reproduced sexually [20]. D. salina, has developed adaptive strategies of survival such as accumulated intracellularly high concentrations of β-carotenes as an antioxidant photoprotector and glycerol as an agent osmorregulator [21]. The cellular chemistry composition is approximately, 50% protein, 20% carbohydrates and 8 % of fatty acids. This species was the first microalgae cultivated industrially for the commercial production of a secondary metabolite, the β-carotene, pigment of high economic value, antioxidant and agent precursor of vitamin A, used as raw material in the pharmaceutical and cosmetic industries [22, 23], as well as the source of the glycerol [24].

Currently, its industrial cultivation has been implemented in many countries, being pioneered in Israel, Australia and the United States of America, where its dry biomass or cell extract is commercialised as a medicinal food. The production of the biomass of D. salina has become a competitive opportunity for emerging businesses that demand natural products traded in large supermarkets and shops pharmaceutical and cosmetic [13, 25-26]. The prospects for the optimization of the cultivation of D. salina with the purpose of obtaining higher biomass productions rich in carotenoids involves, the understanding of those special conditions in which a strain can achieve greater growth; this has given place to a variety of scientific and technological efforts that include the design and implementation of cultivation systems closed as photobioreactors, genetic manipulation of strains, elucidation of the regulatory processes involved in the overproduction of carotenoids [27-31].

Bioprospecting has been a strategy that has allowed the identification of algal specimens with desired attributes (for example, high levels of lipids, growth rates, densities of growth and/or the presence of valuable co-products), although the potential of this strategy would not achieve the level of competitiveness and so are not implemented the latest advances in genetic engineering-improving the biogenesis of lipids and maintenance of culture for obtaining viable strains for different applications -production of metabolites of commercial interest [32, 33]. For that reason, it has been necessary to promote research related to the prospecting of microalgae because it represents a biological resource almost unexplored and exploded. Only 15 species of microalgae, out of 25.000 that have a biotechnological use, have been reported.

The artificial saline of Manaure is located on the coastal marine area of the municipality of Manaure in La Guajira department, Colombia, consisting of a series of interconnected ponds through gates through which flows the water of sea from a reservoir to the ponds of concentration and crystallization [34]. The flow of water in this pond are by the effect of gravity and is controlled by the clogging of the gates that exist between the ponds. The average depth of the ponds varies between approximately 50 and 80 cm according to the rainfall or drought season. This includes an extension of 40.72 Km2 in water mirrors. The aim of this prospective study was to test the effect of stressful conditions-high luminous intensity, high salt concentration and nitrogen deficiency on growth and the production of carotenoids from a strain of D. salina isolated from the artificial of Manaure’s saline.

MATERIALS AND METHODS

Sample collection

The strain of D. salina was isolated from water samples collected with glass jars-previously washed with HNO3 to 10%, rinsed with distilled water and sterilized in the autoclave of the bottom, surface and salt crystals of three artificial ponds distributed in different sites of the saline of Manaure, located at coordinates 11 ° 46' 31.8'' latitude north and 72 °27' 30.5'' longitude west, municipality of Manaure, department of La Guajira (fig. 1).

The samples were enriched with modified Johnsons medium J/1-[35], conserved in a cool box and transferred to the laboratory. The field phase corresponded to the dry season, represented by a high solar radiation (900-µmol. m-2. s-1), the temperature of the surface layer of water approximately 60 °C, the salinity of 4.0 M NaCl with the crystallized bottom and a pH that ranged from 7.55 to 7.77.

Fig. 1: Satellite photography and artificial pond of the saline of Manaure, La Guajira-Colombia (A) Satellite image of the artificial ponds; (B) Artificial pond No. 0811 of Manaure´s saline, La Guajira-Colombia

Isolation of Dunaliella salina of saline of manaure

The purpose of isolation is to obtain non-axenic cultures monoclonal cells of D. salina, using as a selective medium J/1-sterilized. The microscopic observation of different cell forms of genus Dunaliella sp. found in natural water samples collected facilitated the process of isolation from the strain. The culture monoalgae D. salina was carried out with the combination of the two methods: the capillary pipetting and streak plates [31].

Capillary pipetting method

The method used to separate the strains of Dunaliella sp. a drop of water from the natural sample diluted in medium J/1, using a micropipette elaborated with a Pasteur pipette previously sterilized and submerged in ethyl alcohol 70% (Brand Merck KGaA, Darmstadt, Germany). The cells observed by the microscope (Nikon Labophot CF, Melville, NY USA) were "caught" and separated into small droplets of a solution of medium J/1. This operation is repeated several times to obtain individual strains, which were then inoculated in vials of 5 ml of medium J/1 and into different concentrations of NaCl-1.5, 2.5 and 3.5 M-. After 12 d, the algal growth is transfered to Erlenmeyer flasks of 50 to 500 ml with 300 ml medium J/1 with the concentrations of NaCl respectively.

Method of streak plates

This procedure was based on the superficial planting of culture samples (obtained from the previous method) in Petri dishes with agar enriched with medium J/1 with 1.5 and 3.0 M NaCl. Each plate sown was covered with polyethylene plastic, arranged so inverted near a source of daylight at 120 µmol. m-2. s-1 (Feilo Sylvania, Bogotá, Colombia) and incubated at 21-23 °C during 12 d until the outbreak of algal colonies. Subsequently, with a sterile inoculating loop, small portions of algae colonies were removed and mixed in a drop of medium J/1 on a glass slide; then the presence of pure strains of Dunaliella sp was verified. Finally, the identified strains were inoculated in 500 ml Erlenmeyer flasks (Duran Group, Wertheim/Main, Germany) with 300 ml medium J/1 in the same way as in the method of pipetting capillary.

Viability and morphological study

For the taxonomic viability of the cells of Dunaliella sp. used the criteria of morphological descriptions and taxonomic identification keys suggested by [36-38]. The morphological description was based on observations of the microscope.

Culture conditions

Preparation of the culture medium

The medium Johnson modified J/1 was used, specific medium for the growth of microalgae of the genus Dunaliella [35], consisting of a mix of solutions of macronutrients, micronutrients and iron with EDTA. The solution of macronutrients contains in a liter of distilled 1,5 MgCl26H2O, 0.5 g MgSO47H2O, 0.2 g KCl, 0.2 g CaCl22H2O, 1.0 g or 10 mmol of KNO3, 0.043 g NaHCO3 and 0.025 g KH2PO4; iron solution with based on a liter of deionized water contains 189.0 mg Na2 EDTA and 244.0 FeCl3H2O; the solution of micronutrients contains in a liter of deionized water, 62.0 mg H3BO3, 38.0 mg (NH4)6Mo 7O4H2O, 6.0 mg CuSO45H2O, 5.1 mg CoCl26H2O, 4.1 mg ZnCl2, 4.1 mg MnCl24H2O (Brand Merck KGaA, Darmstadt, Germany). Once prepared and sterilized in the autoclave, the nutrient solution was adjusted to pH 8.20 with HCl or NaOH to 0.1%, respectively.

Tests in stressful conditions and non-stressful

The monoclonal cultures of Dunaliella sp. were scaled to 500 ml medium J/1 and maintained to the following culture conditions: (a) conditions non-stressful with medium J/1 to 1.0 M NaCl, 190 µmol. m-2. s-1, 5.0 mmol KNO3, pH 8.2 to test the green phase and ratio of the value of total chlorophyll to carotenoids, and b) stressful conditions with medium J/1 to 4.0 M NaCl, 390 µmol. m-2. s-1, 0.50 mmol KNO3 to prove the reversibility of the red phase and the ratio value of carotenoids to total chlorophyll [39]. The duration of the tests were twenty-one days.

Growth parameters

The population growth de Dunaliella salina in relation to the conditions of the culture was performed using cell count daily and at the same time, with a camera Neubauer (Marienfeld Superior, Cologne, Germany) (cells/ml) on the optical microscope binocular from the first day until the early stage stationary, stage in which it was observed the maximum reversibility of the phases. The cell density, specific rate of growth and doubling time was estimated according to the methodology of [40].

Quantification of production of pigments

The quantification of the production of chlorophyll α and β and total carotenoids of D. salina in function of the culture conditions was carried out with samples of biomass. The extraction of pigments was obtained with acetone 90%. The extract was measured with the absorbance at wavelengths of 480, 647 and 664 nm in a spectrophotometer visible (Genesys™ 20-Thermo Scientific, Basingstoke, Hampshire UK) against a white acetone 90%. The concentration of the pigments of chlorophyll α and β are calculated according to the equation of Jeffrey and Humphrey [41] and for carotenoids, the equation of Strickland and Parson is used [42].

Statistical analysis

Comparisons were made of the cultivation conditions with the production of carotenoids using the model I ANOVA, once checked the assumptions of normality and homoscedasticity to a P-value<0.05 level of significance. The statistical software used for the processing of the data was SPSS Statistics 17.0.

RESULTS AND DISCUSSION

Morphological characteristics of the isolated strain

The strain isolated from the saline of Manaure (La Guajira, Colombia) observed in the optical microscopy (40X) showed morphological characteristics typical of the microalgae Dunaliella salina with red and orange cells, ovoid and/or spherical, with the previous semi round ends and round edges, radially symmetrical; lacks a cell wall polysaccharide rigid thus enabling it to carry out rapid volume changes in response to external changes of the osmotic pressure [43]; chloroplast with a silhouette in the shape of a cup and side lobes well developed which reached the basis of body flagellar; likewise, account with an anterior stigma, diffuse and difficult to distinguish, especially in red cells; a length ranging between 6.1±0.5 to 27.0±1.2 μm and a width between 4.0±0.3 to 20.3±2.1μm (p<0.05).

Stress conditions

It was shown in the effect of conditions under stress on the growth and activity carotenogenic D. salina. The cells experienced a reversal in tonality from green to red phase, which has been observed in other strains of D. salina exposed to conditions of similar culture [36, 44-46].

The increase of the concentrations of carotenoids from D. salina was correlated directly with the gradual increase of the light intensity and salinity, as well as the limitation of the nitrogen source in the growth medium. The implementation of these stress conditions suggests an alternative for improvement in the production of carotenoids based on the simulation of the natural conditions of the stress of this strain [47].

The induction of the carotenogenesis of D. salina is attributed to an evolutionary adaptation that is probably acquired as a response due to the stressful environmental conditions such as extreme temperatures for other microorganisms-25-45 °C, high luminous intensities and salinities between 30-40% of NaCl [48], which favors the proliferation of a small number of species of microorganisms, among these, this strain that pigments the waters with an intense reddish shade, evident features at the time when the samples were taken in the ponds.

These environmental conditions have converted to D. salina a microorganism of interest to the elucidation of physiological processes, mechanisms of adaptation and regulation in the synthesis of carotenoids [49], likewise as a biotic resource of interest when designing and implementing biotechnological applications [12, 29].

The results obtained in the experiments of reversibility of phase as a criterion for the taxonomic location of the isolated strain of D. salina showed an inverse relationship between growth and the production of pigments chloroplastics (table 1, fig. 2 and 3). The conditions of stress-induced the gradual increase of carotenoids reaching the highest concentration the day 20 with a value of 9.67±0.19 µg/ml (p<0.05). In this case is showed that the conditions of stress as high luminous intensity (390 µmol/m2/s), high concentrations of NaCl (4 M) and deficiency of NO3-(0.50 mmol KNO3-) exert a different effect between the optimal growth in relation to the biosynthesis and accumulation of carotenoids, physiological capabilities observed in D. salina strains grown in the laboratory in relatively similar conditions [50].

Table 1: Production of pigments of Dunaliella salina in relation to stressful conditions and not stressful

Tests

Time

(days)

MCDa

ACTChb

(µg/ml)±SD

ACTCac

(µg/ml)±SD

Reason

ChT/CaT

Reason

CaT/ChT

Reversibility red phase to green 1 0.8±0.26 3.78±0.13 0.38±0.02 9.95±0.80 0.10±0.007
10 3.12±1.09 6.15±0.18 0.9±0.03 6.83±0.36 0.15±0.007
20 1.9±0.2 4.25±0.45 1.54±0.08 2.76±0.25 0.36±0.034
Reversibility green phase to red 1 0.8±0.21 2.16±0.31 0.43±0.017 5.02±0.58 0.20±0.022
10 2.04±0.49 4.3±0.19 6.5±0.32 0.66±0.06 1.51±0.133
20 0.78±0.1 2.95±0.13 9.67±0.19 0.31±0.02 3.28±0.207

aMCD: Average Maximum cell density (106 cel. ml-1)±SD* bACTCh: Average concentration of total chlorophyll cACTCa: Average concentration of total carotenoids *SD: Standard Deviation

The variability of the content of lipophilic antioxidants (carotenoids) in D. salina strains are found related to the activities of the antioxidant enzymes superoxide dismutase, catalase and peroxidase, as well as cell, production of malonaldehyde and free radicals of D. salina in response to the exposure of electromagnetic radiation of UV-B, commonly in conditions of high solar irradiation and limitation of nitrogen and high concentrations of NaCl [29]. In cultures with non-stressful conditions- optimal values of NO3-, salinity (9%) and lighting 190 µmol. m-2s-1- the MCD and the production of total chlorophyll for the volume as a function of time of the test is greater than the reversibility of phase green to red. The comparison between the values of cell densities and the production of total chlorophyll per volume of D. salina were obtained with the experiment with non-stressful conditions with regard to the stressful ones presented significant differences (p<0.05, fig. 2).

This is explained because of the concentration of N in the culture medium is directly proportional with the growth of the strain in accordance with the production of total chlorophyll that is an indicative of a photosynthetic rate high and cell growth [49]. The observations in the microscope revealed that from day 8 during the exponential phase cells showed higher mitotic activity, great size and intense green color, which suggests that adequate conditions of lighting, salinity and nutrients, studied in this experiment, which allows the strain to develop itself in physiological favorable conditions [49, 50].

Fig. 2: Production of total chlorophyll of D. salina in relation to stressful and non-stressful cultures


Fig. 3: Production of total carotenoids of D. salina in relation to stressful and non-stressful cultures

The significant difference (p<0.05) between the average values of total carotenoids by volume cultures in the experiment of reversibility of phase green to red with 9.67±0.19 µg/ml in contrast to the reversibility of red phase to green with 1.54±0.08 µg/ml, shows the effect of the stressor factors already described on D. salina, which it is shown at the start of the experiment the dark green cells and cells of a smaller size than the cells of reddish color with greater size at the end of the experiment, followed by a significant decrease in the MCD in contrast with the experiment with factors non-stressful.

This response to the increase in total carotenoids content is in first place, depending on the decrease of the nitrogen source in the culture medium that accompanied with an increase of pH promotes the precipitation of phosphate salts causing a state of deficiency, in response to this, cells decrease the activity of the photosystem II and photosynthesis [51]; in second place when an algal strain is influenced by a strong radiation can cause damage in the centers of reaction and in primary response to this factor promotes the accumulation of carotenoids with a possible role photo protector [43, 52, 53].

The salinity effect

The concentration of salinity affects the kinetics of growth as well as the metabolic processes of the algal cells, which is a clear inverse correlation between the MCD and the salinity (4 M NaCl) isolated from D. salina in the experiment with stressor cultures. This condition has been demonstrated in studies with strains of D. salina [36, 44, 55]. This stress in salinity restricts the capacity of D. salina to acquire the necessary sources for its growth and productivity.

In general, the carbon fixation by photosynthesis is delayed in cells exposed to a variety of stresses. In spite of what has said above, in cells of D. salina there is a stimulation in the rate of acquisition of inorganic carbon and it has been determined the presence of a carbonic anhydrase adapted to high salinities [56]. A characteristic response of D. salina to stress by salinity is the adjustment in the intracellular concentration of glycerol by regulating the flow of carbon between the production of starch in the chloroplast and the synthesis of glycerol in the cytoplasm.

Thus, the flow of carbon is channelled from starch toward the glycerol with a concomitant increase in the biosynthesis of plastidic isoprenoids, this is, an increase of carotenoids [57-59]. The microscopic observations showed a contrast in the average of the cell size when the microalgae were exposed to the osmotic changes. After a change, hyperosmotic cells increased their volume gradually depending on the concentration of NaCl of the culture medium and of the exposure time (average cell volume of approximately 1380.36±0.03 µm3, 1.6 times greater than those of logarithmic phase) (fig. 4). These observations coincide with the results presented by [46] above all, when the cells of D. salina exposed to conditions of osmotic stress reach concentrations significantly homogeneous of carotenoids.

Fig. 4: Reversibility of phase green to red of Dunaliella salina. (A) Isolated from D. salina; (B) D. salina by changing its tonality (day 10); (C) D. salina in late stationary phase (day 20). The scale bar represents 10 µm

CONCLUSION

The conditions of stress such as high luminous intensity (390-µmol/m2/s), high concentrations of NaCl (4 M) and deficiency of NO3-(0.50 mmol KNO3-) exert a different effect between the population growth and the biosynthesis and intracellular accumulation of carotenoids from the D. salina strain isolated from Manaure´s saline.

Stress conditions evaluated in cultures such as N source limitation, NaCl concentration increase and light intensity induced the carotenogenesis of the D. salina strain isolated from the Manaure´s saline.

The isolated D. salina strain exhibits phenotypic traits that allow it to survive in extreme conditions such as extreme temperatures for other microorganisms less than 25 to 45 °C, high luminous intensities and salinities between 30-40% NaCl.

The biochemical and metabolic characteristics exhibited by D. salina due to the conditions of the studied cultures suggest an alternative of improvement in the production of carotenoids in biotechnological and industrial terms.

The D. salina strain isolated from Manaure artificial saline could represent a biological resource of importance for the pharmaceutical, cosmetic and nutraceutical industry due to its great capacity for lipophilic antioxidants, enzymes, biopolymers or compatible solutes; in addition its physiological properties and ease of cultivation in closed systems like photo bioreactors make them an attractive and economically viable technological alternative for commercial exploitation [60, 61].

CONFLICTS OF INTERESTS

All authors have none to declare

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How to cite this article

  • Gallego-Cartagena Euler, Castillo-Ramírez, Martínez-Burgos Walter. Colombian strain of Dunaliella salina as a source of metabolites of high commercial values. Int J Pharm Pharm Sci 2017;9(4):32-37.

About this article

Title

COLOMBIAN STRAIN OF DUNALIELLA SALINA AS A SOURCE OF METABOLITES OF HIGH COMMERCIAL VALUES

Keywords

Dunaliella salina, Carotenoides, Saline of Manaure, Stressful conditions, Biotechnological purposes

DOI

10.22159/ijpps.2017v9i4.15876

Date

14-02-2017

Additional Links

Manuscript Submission

Journal

International Journal of Pharmacy and Pharmaceutical Sciences
Vol 9, Issue 4, 2017 Page: 32-37

Online ISSN

0975-1491

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Authors & Affiliations

Gallego Cartagena Euler
Faculty of Environmental Science, Universidad de La Costa, Barranquilla 3599481, Atlántico, Colombia
Colombia

Castillo RamÍrez Margarita
Faculty of Environmental Science, Universidad de La Costa, Barranquilla 3599481, Atlántico, Colombia
Colombia

MartÍnez Burgos Walter
Faculty of Enginering, Universidad del Norte, Barranquilla 3509509, Atlántico, Colombia
Colombia

 

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