Natl. Acad. Sci. Lett. DOI 10.1007/s40009-012-0038-6
RESEARCH ARTICLE
Isolation and Characterization of Dunaliella Species from Sambhar Lake (India) and its Phylogenetic Position in the Genus Dunaliella Using 18S rDNA Pooja Sharma • Varsha Agarwal • M. Krishna Mohan Sumita Kachhwaha • S. L. Kothari
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Received: 22 December 2011 / Accepted: 19 May 2012 Ó The National Academy of Sciences, India 2012
Abstract High b-carotene producing microalga Dunaliella, isolated from Sambhar Lake, India, was identified, characterized and analyzed for its phylogenetic position in the genus Dunaliella. It was characterized on the basis of morphology and 1.7 Kb PCR product produced using the primers MA1 and MA2 which is specific to Dunaliella. Four stress parameters were used for characterization i.e. NaCl concentration, temperature, light and nitrogen concentration. On these parameters total cell number, specific growth rate and total carotenoids/chlorophyll a ratio was analyzed. 1.7 M NaCl, 26 °C temperature, diurnal light and 4.9 mM N-concentration were identified as optimum conditions for growth whereas 3.5 M NaCl, 37 °C temperature, continuous light and without nitrogen source were identified as optimum conditions for carotenoid production. Highest cell number (19.6 9 105 cells/ml) and specific growth rate (0.505 division per day) were observed at 1.7 M salinity whereas highest total carotenoids/chlorophyll a ratio (1.57) was observed at 3.5 M salinity. 17 sequences of 18S rDNA of Dunaliella, taken from NCBI nucleotide database, were used for phylogenetic analysis. A bootstrap consensus tree was constructed using neighbour-joining method.
P. Sharma V. Agarwal S. Kachhwaha S. L. Kothari (&) Centre for Converging Technologies (CCT), University of Rajasthan, Jaipur 302 004, India e-mail:
[email protected] M. Krishna Mohan Birla Institute of Scientific Research, Statue Circle, Jaipur 302 001, India S. Kachhwaha S. L. Kothari Department of Botany, University of Rajasthan, Jaipur 302 004, India
Keywords 18S rDNA Carotenoids Dunaliella NCBI Stress condition
Introduction Dunaliella, is a chlorophycean alga which is unicellular, biflagellate and devoid of cell wall. It is ovoid, spherical or ellipsoid with size varying from 16 to 24 lm in length and from 9.5 to 13.3 lm in width [1]. It has remarkable capacity to grow and adapt to media ranging in salinity from 50 mM to 5 M NaCl. The major means of osmoregulation is through production of intracellular glycerol [2] at a concentration that is proportionate to the external NaCl concentration [3]. It is an important organism that can accumulate high amounts of b-carotene up to 10 % of its dry biomass [4] resulting in orange-colored growth [5]. b-Carotene is marketed as a food coloring agent, provitamin A in food and animal feed, an additive to cosmetics, for multivitamin preparation and, in the past decade, for its purported anti-oxidant properties [6]. Several industrial production plants of Dunaliella salina are operating in Australia, Israel, USA, China and Japan [7], where annual production is up to 1,200 tonnes dry weight [8]. Various species of Dunaliella are known, which have different requirement for growth, salinity tolerance and carotenoid production. Identification of Dunaliella is ambiguous when only morphological characteristics or physiological characteristics are considered, since it has diverse morphological and physiological characteristics depending on the conditions of growth. Molecular identification is a useful tool to distinguish between inter- and intra-specific species with similar morphology [9]. It is well established that the type and degree of nutrient limitation result in dramatic changes in the cellular chemical
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composition of phytoplankton [10]. Under favourable growth conditions, Dunaliella contains only those pigments (carotenoids and chlorophyll) that are necessary for photosynthesis [11]. In contrast, under stress conditions, it produces high amount of carotenoids (b-carotene). The present study was aimed to isolate the chromosomal DNA for amplification of nuclear-encoded small subunit ribosomal RNA gene (18S rDNA) segment in order to confirm the saline lake microalga as Dunaliella, because this technique has been found to be a great help in the identification of Dunaliella [12]. Morphological identification of Dunaliella using microscopy and physiological characterization (total cell number, specific growth rate and total carotenoids/chlorophyll a ratio) at various parameters (NaCl concentration, temperature, light and nitrogen concentration) was also examined. To the best of our knowledge this is the first report of phylogenetic analysis of Dunaliella sp., from Sambhar lake (Rajasthan, India), among various species of Dunaliella using 18S rDNA sequence.
Amplification and Sequencing of 18S rDNA Gene The primers, MA1 (50 -CGG GAT CCG TAG TCA TAT GCT TGT CTC-30 ) and MA2 (50 -CGG AAT TCC TTC TGC AGG TTC ACC-30 ) [9] procured from Genei, Bangalore (India) were used to molecular identification of D. salina [12] isolated from Sambhar Lake. PCR reaction was performed in a total volume of 25 ll, containing 50 ng of chromosomal DNA in TE buffer pH 8.0 and 25 ng of each primer. The amplification was performed in Mastercycler pro, Eppendorf. One cycle consisted of 45 s at 94 °C, 45 s at 60 °C and 2 min at 72 °C (with 15 s time gradient). PCR product was resolved in 1.2 % agarose gel along with 1.0 Kb marker. Amplified 18S rDNA was purified using Gel Extraction Kit (Genei Bangalore, India). PCR product was sequenced from Sequencher Tech Pvt. Ltd., Ahmedabad, India and submitted to National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm. nih.gov) gene data bank. Phylogenetic Analysis
Materials and Methods Isolation of Dunaliella Water samples were collected from Sambhar Lake, Rajasthan, India. Samples were microscopically examined for the presence of Dunaliella. Bacterial and fungal contaminations were eliminated by adding the mixture of ampicillin, streptomycin, tetracycline (200 lg/ml) and bavistin (Carnbandazin powder) (50 lg/ml). Pure culture was obtained using streaking technique. Culture Conditions The isolated alga was cultured on medium containing 1.7 M NaCl, 7.3 mM MgCl26H2O, 2.0 mM MgSO47H2O, 2.6 mM KCl, 2.3 mM CaCl2, 4.9 mM KNO3, 4.9 mM Tris Buffer, 8.5 lM EDTA di-Na salt, 0.58 lM ZnCl2, 19.0 lM H3BO3, 0.12 lM CoCl2, 0.46 lM CuCl2, 6.3 lM MnCl2, 9.2 lM FeCl3, 1.4 mM KH2PO4. To avoid precipitation, FeCl3 and KH2PO4 were autoclaved separately. Dunaliella was grown in 250 ml Erlenmeyer flask containing 100 ml medium at 26 ± 1 °C temperature, manually shaken twice in a day and a 16:8 h (light/dark cycle) photoperiod with 50 lmol m-2 s-1 illumination provided by cool white fluorescent lamps (Philips, India). Sub-culturing of Dunaliella was carried out after every 20 days.
Phylogenetic analysis was based on 17 sequences of 18S rDNA of Dunaliella. The 18S rDNA sequences were taken from NCBI nucleotide database (Table 1). The distance matrix and neighbour-joining (N-J) method were used to construct the tree. Branches corresponding to partitions reproduced in less than 50 % bootstrap replicates (total 1,000 replicates) were collapsed. The evolutionary distances were computed using the Kimura 2-parameter method [14] and were in the units of the number of base substitutions per site. There were a total of 1,687 positions in the final dataset. Evolutionary analyses were conducted in Molecular Evolutionary genetics Analysis software (MEGA5) [15]. Growth Measurement Samples for growth measurement were taken 11 times during the 20 days incubation period and counted using a Neubauer haemocytometer. Specific growth rate l (division day-1) was estimated using standard formula l ¼ 3:3ðlogN2 logN1 Þ=t where N2 is the optical density of final reading; N1 is the optical density of Initial reading; t is the time interval (in days). Pigment Estimation
Isolation of Genomic DNA DNA was isolated using TEN buffer (Tris 10 mM pH 8.0, EDTA 10 mM, NaCl 150 mM) method [13].
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Chlorophyll a and b-carotene were extracted from an algal pellet with acetone, diluted to 80 % acetone with water (v/v) and assayed according to Lichtenthaler et al. [16].
Natl. Acad. Sci. Lett. Table 1 18S rDNA nucleotide sequences of Dunaliella species from NCBI database to analyze phylogenetic position of the isolate from Sambhar Lake (in Bold) S. no.
Species of Dunaliella (18S rDNA sequences)
Gene-Bank accession number
1
D. tertiolecta strain SAG 13.86
EF473737.1
coloration due to characteristic accumulation of b-carotene. Cells growing at 1.7 M NaCl concentration in the medium are shown in Fig. 1a. Cultures growing in the medium with 0 and 4.9 M nitrogen concentration are shown in Fig. 1b. Molecular Identification of Dunaliella
8
D. salina strain CCAP 19/30
EF473749.1
9
D. bardawil strain I2
JF831045.1
Genomic DNA was resolved (Fig. 1c) and amplified 1.7 Kb PCR product of 18S rDNA gene was obtained (Fig. 1d). The 18S rDNA sequence has been submitted to NCBI gene bank (Gene-Bank Accession No. HQ843776.1). The amplified 1.7 Kb PCR product of 18S rDNA was similar in size to the previously amplified PCR product [9, 17, 18]. There is not a single report of molecular identification of Dunaliella, isolated from Sambhar Lake.
10 11
D. salina strain JR102 Dunaliella sp. (Sambhar lake India)
EU589200.1 HQ843776.1
Phylogenetic Analysis
2
D. salina strain N5
JF83104.1
3
D. salina strain Dsge
EF473731.1
4
D. tertiolecta strain CCAP 19/27
EF473747.1 EU589199.1
5
D. salina strain JR101
6
D. salina strain SAG 42.88
EF473740.1
7
D. tertiolecta strain Dtsi
EF473729.1
12
D. salina (Gujarat India)
EF195157.1
13
D. salina
AF506698.1
14
Dunaliella sp. ABRIINW-G3
GU984571.1
Results and Discussion
In phylogenetic tree, the branch lengths were proportional to the amount of inferred evolutionary change. The bootstrap consensus tree was constructed from N-J method using 18S rDNA (Fig. 2). Pair wise distance was calculated and overall average came to be 0.008 with 0.001 standard error. The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The percentage of replicate trees in which the associated strain clustered together in the bootstrap test has been shown next to the branches [19]. As seen in Fig. 2, 10 species of Dunaliella i.e. D. tertiolecta strain SAG 13.86, D. salina strain N5, D. salina strain Dsge, D. tertiolecta strain CCAP 19/27, D. salina strain JR101, D. salina strain SAG 42.88, D. tertiolecta strain Dtsi, D. salina strain CCAP 19/30, D. bardawil strain I2 and D. salina strain JR102 were clustered together. In another cluster, Dunaliella sp. isolated from Sambhar Lake, Rajasthan was found to be similar with D. salina isolated from Central Salt and Marine Chemicals Research Institute (CSMCRI), Bhavnagar, Gujarat. Dunaliella sp. ABRIINW-G3, Dunaliella sp. ABRIINW U1/1, D. salina isolate I3 and D. salina isolate B32 appeared as individual entity. In earlier studies, genetic or phylogenetic variations among D. salina strains were analysed using RAPD [13] and ITS rDNA sequences [1, 20]. Nakayama et al. [21] studied phylogenetic analysis between chlamydomonadales and chlorococcales inferred from 18S rDNA sequence data.
Morphological Identification of Dunaliella
Effect of Salinity
Pure cultures of Dunaliella showed spherical to ovoid, biflagellate cells in vibrating motion under light microscope. Flagella were approximately equal to length of the cell. Under stress conditions, cells showed a yellow-orange
Maximum cell number was observed at 1.7 M NaCl concentration i.e. 19.625 9 105 cells/ml on 8th day of incubation (Fig. 3a), while culture containing 4.5 M concentration turned white within 1 day after inoculation.
15
Dunaliella sp. ABRIINW U1/1
FJ164062.1
16
D. salina isolate I3
HQ735295.1
17
D. salina isolate B32
HQ735296.1
The spectra were analyzed using UV-1800 spectrophotometer, Shimadzu (Japan). Stress Parameters Four stress parameters-salinity, temperature, light and N-concentration were considered to find out capacity to accumulate carotenoids or b-carotene of Dunaliella cells. For salinity stress, seven concentrations of NaCl were used (0.5, 1.0, 1.7, 2.0, 2.5, 3.5, and 4.5 M). For temperature stress, two temperatures were considered (26 and 37 °C). For irradiance stress, effect of continuous light over diurnal light was analyzed. For stress of N-concentration, three concentrations of KNO3 were considered (0, 2.5 and 4.9 mM). All the stress experiments were conducted for 20 days. The samples were taken after every 48 h and analyzed for total cell number, specific growth rate and total carotenoid/chlorophyll a ratio.
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Natl. Acad. Sci. Lett. Fig. 1 a Dunaliella cells growing at 1.7 M NaCl concentration (100X). b Yellow colored culture of Dunaliella showing b-carotene production without nitrogen source (stress condition), whereas green colored culture of Dunaliella at 4.9 mM N-concentration were showed (growth condition). c Genomic DNA isolated from Dunaliella in 0.8 % agarose gel with Lambda DNA-Hind III digest marker. d PCR amplification of 1.7 Kb 18S rDNA gene in 1.2 % agarose gel with 1.0 Kb Marker, Biolabs
Fig. 2 Using 18S rDNA sequences, the phylogenetic tree was constructed by neighbourjoining method to analyze phylogenetic position of Dunaliella, isolated from Sambhar Lake. The degree of support for internal branches of the tree was assessed by bootstrap method
D. tertiolecta strain SAG 13.86 D. salina strain N5 D. salina strain Dsge D. tertiolecta strain CCAP 19/27 72
D. salina strain JR101 D. salina strain SAG 42.88 D. tertiolecta strain Dtsi D. salina strain CCAP 19/30
76
D. bardawil strain I2 D. salina strain JR102 Dunaliella sp. (Sambhar lake India)
67
98
63
D. salina (Gujarat India) D. salina Dunaliella sp. ABRIINW-G3 Dunaliella sp. ABRIINW U1/1 D. salina isolate I3 D. salina isolate B32
Log phase of culture was observed up to 10–12 days after the cell number decreased. 1.7 M concentration showed highest specific growth rate (0.50522 division per day) on 5th day (Fig. 3b). Specific growth rate decreased on 1st day at 0.5, 1.0, 2.0, and 3.5 M NaCl-concentrations thereafter it increased. Maximum total carotenoids/chlorophyll a (car/ chl a) ratio was observed at 3.5 M salinity (1.57) (Fig. 3c).
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The optimum salt concentration for growth was 1.7 M and for carotenoid production was 3.5 M. In previous studies, optimum growth was observed at 1.0 M [17], 1.7 M [22], 2.0 M NaCl [23–25]. Different growth patterns, exhibited by geographically distinct strains, confirm the hypothesis that Dunaliella have ability to tolerate a wide range of salinities [22]. Maximum
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Fig. 3 Effect of NaCl on a cell number. b Specific growth rate per day. c Total car/chl a
Fig. 4 Effect of temperature on a cell number. b Specific growth rate per day. c Total car/chl a
growth rate of Dunaliella from Sambhar lake was 0.50522 day-1 whereas growth rate of D. salina from Mexico showed 0.28 day-1 [7]. In previous studies, salinity for maximum carotenoid production was 4.0 M [24, 25]. Growth of isolated Dunaliella was observed in the range of 0.5-3.5 M NaCl concentration, whereas in other studies range was 0.5–5.5 M [17], 1.5–4.4 M [25], 1.7–6.0 M [22], 0–3.5 M [26], 1–5 M [24]. Effect of Temperature At 26 °C maximum cell number (19.625 9 105 cells/ml) and growth rate (0.50522 division per day) were observed. At 37 °C maximum cell number was 11.125 9 105 on 16th day (Fig. 4a) and specific growth rate was 0.403 on 7th day (Fig. 4b). Car/chl a ratio increased with temperature and rose up to 0.93 (Fig. 4c). Higher growth rate, cell number and chlorophyll content in Dunaliella were observed at 26 °C than 37 °C, whereas high carotenoids were observed at 37 °C and ratio of car/chl a was 0.93. Maximum specific growth rate was observed at 26 °C that is 0.5 division day-1 whereas specific growth rate of D. salina CONC-001 chile, D. salina CONC-006 chile, D. salina CONC-007 chile, D. salina Maxican, D. salina Chinese, D. salina Australian and D. bardwil Isreal was 0.9, 0.6, 0.7, 1.1, 1.2, 0.9, and 1.0 division day-1, respectively at 26 °C and 40 lmol m-2 s-1 light intensity [6]. Garcia et al. [7] showed that higher cell number of D. salina Mexico, was observed at 22 °C than 38 °C temperature.
Effect of Light The effect of continuous and diurnal light on cell number and specific growth rate was observed (Fig. 5a, b). Maximum cell number at continuous and diurnal light was 15.0 9 105 and 19.625 9 105 cells/ml, respectively on 8th day of inoculation. Specific growth rate at continuous and diurnal light was 0.44081 (3rd day) and 0.50522 (5th day) respectively. Higher carotenoid production was observed at continuous light than diurnal light and a ratio of 0.72 on 17th day (Fig. 5c). Light plays an important role in carotenoids production. b-carotene globules protect the cell against injury by high intensity irradiation under limited growth conditions by acting as a screen to absorb excess irradiation [4, 25]. Enhanced carotenoid production (1–4 times) was observed in continuous light than diurnal light while more growth and chlorophyll contents were observed in diurnal light than continuous light. Effect of N-Concentration As concentration of nitrogen decreased, cell number and specific growth rate decreased and car/chl a ratio increased. In medium without nitrogen source, cell number (7.875 9 105 cells/ml) was less than half at 4.9 mM concentration (Fig. 6a). Maximum cell number at 2.5 mM concentration was 16.625 9 105 cell/ml on 8th day. Specific growth rate at zero nitrogen was also half than
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Fig. 5 Effect of light on a cell number. b Specific growth rate per day. c Total car/chl a
Fig. 6 Effect of nitrogen concentration on a cell number. b Specific growth rate per day. c Total car/chl a
4.9 mM concentration (Fig. 6b). Highest car/chl a ratio was 0.78 at zero nitrogen on 10th day (Fig. 6c). Dunaliella produces high concentration of b-carotene at any stress conditions. Nitrogen is primary requirement for the growth of any living cells. Under minimum concentration of nitrogen, Dunaliella consider it as a stress condition and produces high amount of carotenoids. When nitrogen is absent in medium, highest car/chl a ratio is observed. As the nitrogen concentration increases in the medium, cell number and specific growth rate also increases. Similar results were showed by Pisal et al. [25]. In conclusion, on the basis of morphology [27], 1.7 Kb PCR product and phylogenetic analysis, this species may be D. salina. The parameters for optimum growth and carotenoid production have been standardized which could be used for mass cultivation of Dunaliella producing bcarotene.
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9. Acknowledgments Pooja Sharma and Varsha Agarwal thank University Grants Commission (UGC) and Council for Scientific and Industrial Research, New Delhi, respectively for the award of SRF.
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