Morphological characteristics of the newly isolated strains
The halotolerant microalgae isolated from the salt pans of Goa were identified as D. salina and D. sp, which vary in cell shape and size from each other (Fig 2 a,b) Both the strains are unicellular and biflagellate. The stigma of D. sp. species is prominent, stick shaped, small and found on the anterior side of the cell, whereas the stigma of D. salina is diffused. In D. salina, carotenoids accumulated in the inter-thylakoid spaces as oil droplets (Fig 2a). In the initial stage, the carotenoids were formed at the periphery of the chloroplast and then spread throughout the organelle as the carotenoid content increases. On the other hand, Dunaliella sp. accumulated carotenoids as a single droplet near the stigma (Fig 2b).
The cell shape, size and colour were found to vary between the two Dunaliella sp. During the culture period, D. sp remained as green, oval shapes, while D. salina slowly changed from green, round shapes to reddish-orange, oval ones. The cell size of D. salina measured approximately 12–18 µm in diameter, with flagellar length of 17–21 µm, whereas D. sp measured 2–6 µm diameter, with flagellar length of 5–10 µm.
The phylogenetic analysis of the ITS region showed that both the species isolated from the salt pans of Goa formed two groups. (Fig 3). The first group was comprised of Dunaliella sp and the isolated species Dunaliella sp G1 clustered with this group. The Dunaliella salina formed the second group and the Dunaliella salina G2 was clustered with this group. The Blast results revealed that isolate Dunaliella salina G2 (Accession No: MW218509) obtained from Batim showed 100% identity with D. salina strain NUAC09 and the isolate Dunaliella sp G1 (Accession No: MW218512) obtained from Ribander showed 99% identity with Dunaliella sp MBTD-CMFRI-S122.
Effect of salinity on algal biomass and pigment production
Both D. salina and D. sp showed an inverse relationship with salinity, where the cell numbers decreased with increase in the NaCl concentration (Fig. 4). D. salina reached a maximum cell density of 11.82 × 106 cells/ml at 0.75 M NaCl concentration, whereas D. sp showed a maximum cell density of 18.76 × 106 cells/ml at 0.5M salinity. Both the strains showed no growth at higher salinity (3M). According to this result the optimum salinity for the maximum growth of D. salina and D. sp was 0.75M NaCl and 0.5M NaCl, respectively. The growth rate of D. sp differed significantly between the cultures exposed to different NaCl concentrations (P≤0.01), In contrast, the growth rate of D. salina grown at different NaCl concentrations was not statistically significant (P≥ 0.05). Both the species accumulated low amount of carotene in their cells under high salinity.
Effect of light intensity on growth and pigment production
The growth curve of D. salina and D. sp exposed to different light intensities of 100, 200, 500,1000 µmol m-2 s-1 is summarized in Fig 5. Under higher light intensities, Dunaliella sp. showed maximum cell growth. Upon exposure to a high light intensity of 1000 µmol m-2 s1 and a low intensity of 100 µmol m-2 s-1, D. salina achieved a maximum cell density of 22.35 ×105 cells/ml and a low cell density of 13.25 ×105 cells/ml, respectively. The maximum cell density (10.03 ×105cells/ml) of D. sp obtained at a light intensity of 1000 µmol m-2 s-1 was comparatively lower than D. salina. The observed variation in the growth rate of D. sp exposed to different light intensity was statistically significant (P ≤0.05) compared with D. salina (P ≥0.05). This growth level was obtained between 15 and 21 days of the experiment. The growth curves show a large increase in cell density after the 7th day (log phase) of inoculation.
The TLC revealed the presence of four prominent fractions of pigments including chlorophyll a, chlorophyll b xanthophyll, β-carotene and phenolic compounds in both D. salina and D. sp. Chlorophyll a, chlorophyll b and β-carotene were the dominant pigments with Rf values 0.14, 0.11 and 0.87, respectively (Fig. 6a). The total chlorophyll and total carotenoid contents of D. salina and D. sp exposed to four different light intensities (100, 200, 500 and 1000 µmol m-2 s-1) are depicted in Table 1. Increased light intensities resulted in decreased total chlorophyll content but, increased total carotenoids in both the Dunaliella species. The observed variation in the total carotenoid production by D. sp and D. salina exposed to different light intensities was not statistically significant (P = 0.059>0.05). However, the total chlorophyll accumulation was statistically significant between the two species (P<0.001)
The total carotenoid content of D. salina ranged between 7.9 and 36.6 pg. cell−1), and chlorophyll concentration ranged between 5.99 and 9.70 pg. cell−1). In D. sp maximum concentration of chlorophyll (12.8–19.9 pg. cell-1) as well as carotene content (2.8–4.0 pg. cell−1) is obtained (Table 1). As per One-way ANOVA, the cellular content of total carotenoids was found to vary significantly in two species under different light intensities (P<0.001). Chlorophyll and β-carotene were the dominant pigments present in the algal strains. HPLC-DAD was used to measure the composition of major carotenoids, namely zeaxanthin, lutein, neoxanthin, β-carotene, chlorophyll a, b, c, and violaxanthin, in both the Dunaliella species in response to the four different light intensities. Fig 6b, shows HPLC chromatogram of the pigments extracts from the two Dunaliella species grown under the light intensities of 1000 µmol m-2 s-1. It’s clear that, in Dunaliella salina β-carotene dominates the carotenoid composition. Dunaliella sp. produced a higher relative amount of lutein and zeaxanthin under high light stress compared with D. salina indicates the important role of these pigment in Dunaliella sp. photoprotection.
D. salina exposed to 1000 µmol m-2 s-1 light produced relatively a higher amount of zeaxanthin (0.20 mg/ml) than Dunaliella sp. showing the important role of zeaxanthin in D. salina for photoprotection. At the same light intensity, D. sp showed considerably higher concentration of lutein (1.08 mg/ml) than D. salina.
The major characteristic difference between these two species was their ability to accumulate β-carotene (Fig 7a). The β-carotene content increased with increasing light intensity in both the species. Under the higher light intensity of 1000 µmol m-2 s-1, the β- carotene content was 4.28 mg/ml and 12.49 mg/ml in Dunaliella sp and D. salina, respectively. Both the Dunaliella species showed a similar response to increasing light by increasing the β-carotene accumulation in the cell. D. salina produced maximum β-carotene than Dunaliella sp. under the four different light intensities examined. We found that the ability of β-carotene production in D. salina much higher than in Dunaliella sp. under light stress.
Fig. 7b, shows the effect of light intensity on the lutein production of the two species. The response of both the species towards increasing light intensities and accumulation of lutein varied considerably among different strains. The lutein content in D. salina did not vary considerably under the four different light intensities. In D. salina, the lutein concentration was inversely proportional to light intensity. D. salina, showed maximum accumulation of lutein content under light intensity of 100 µmol m-2 s-1. The maximum Lutein content was increased under 1000 µmol m-2 s-1 of light intensity in Dunaliella sp. In Dunaliella sp the lowest lutein content at 100 µmol m-2 s-1 was obtained.
The cellular content of zeaxanthin in the two species grown under different light intensities is shown in Figure 7c. Dunaliella sp. strain accumulated the highest amount of zeaxanthin under higher light intensity, whereas D. salina accumulated the lowest. Dunaliella sp. contains a higher cellular content of zeaxanthin at light intensity of 1000 µmol m-2 s-1, whereas D. salina accumulates higher zeaxanthin content under 100 –200 µmol m-2 s-1. Under different light intensities the accumulation of zeaxanthin was significantly different among strains.
Figure 7 (d) shows that neoxanthin content in two Dunaliella sp. Both species show a different response to different light intensities. D. sp had the highest pigment concentration under high light intensity.