The S. quadricauda CASA-CC202 (KM250077) cultures were grown in optimized conditions as described by Anand and Arumugam, 2015. The algal cells were grown at 25 ± 2°C with 14:10 h light–dark period. The flasks were gently shaken twice daily to avoid adherence of the cells to the walls, and there was no air and CO₂ was supplemented during the experimental period. The salt stress was induced when the cells grown to its log phase harvested by centrifugation (10000 rpm for 10 min), and the pellets were washed with double distilled water. Then the pellets were transferred to the stress media with different NaCl concentration (10–50mM), and subsequently, the nitrogen stress was induced (Sulochana and Arumugam, 2016).

The experimental algal cells were prefixed with 4% paraformaldehyde, subsequently, the fixed cells were suspended with sensitization solution composed of 0.1M Tris-7.5, 0.15M NaCl, and 0.1% Triton X-100 for 30 min. Sensitized cells were washed twice with 0.1 M Tris-7.5 with 0.1% Triton X-100 in the same tube. MLDP polyclonal antibody was added in 1: 1000 dilution ratio, and incubated for 3 h in rocker at 4°C. Cells were washed thrice with wash solution followed by incubation with secondary antibody (anti-rabbit antibody) conjugated with FITC for two more hours. The cells were washed three times and suspended in 1× PBS and viewed under a confocal microscope.

At every 5-day interval, the samples were drawn aseptically from the stressed and control flasks, and the growth was measured spectrophotometrically at 540 nm. Cell number, a direct microscopic enumeration, was performed at every 5-day intervals as described in Anand and Arumugam (2015). The specific growth rate was measured, number of generation (doubling time), that occurs per unit of time in an exponentially growing culture. The phase of growth was carefully determined, and the specific growth rate was obtained using the following equation (Guillard and Ryther, 1962).

N_t = number of cells at the end of the log phase; N₀ = number of cells at the start of log phase; T_t = final day of log phase; and T₀ = starting day of log phase. If T expressed in days from the growth rate (μ) can be converted to division or doubling per day (K) by dividing (μ) by the natural log of 2(0.6931). In order to determine the dry biomass yield, 10 ml samples were filtered on to pre-dried and weighed GF/C fiber filters every third day of intervals. The filtered biomasses were oven dried overnight at 60°C along with filter paper, and reweighed using an analytical balance. The dry biomass of Scenedesmus under salt stress condition was expressed in grams per liter.

Total lipid was estimated by Folch et al. (1957) method for different NaCl-treated S. quadricauda. In brief, 5 ml of culture was taken from 400 ml sample in a 1000 ml Erlenmeyer flask. The biomass was collected by centrifugation at 10000 rpm for 10 min and suspended in 6 ml of chloroform:methanol (2:1) for 5 h, then 2 ml of 0.9% saline was added and mixed vigorously, and incubated for 12 h. From the lipid-containing lower phase, 0.5 ml was pipetted out and transferred to a fresh 2 ml Eppendorf tube and allowed for evaporation. After that, 0.5 ml of concentrated sulfuric acid was added and mixed well, and the tubes were kept in a water bath for 15 min and cooled to room temperature. From that, 0.2 ml was pipetted out and transferred to the fresh test tube containing 5 ml of vanillin reagent, was mixed well, and incubated for 20 min at room temperature. After incubation, the appearance of pink color indicates the presence of lipid, and then the sample was read at 520 nm.

The total photosynthetic algal sample was taken and centrifuged at 8000 rpm for 10 min and the supernatant was discarded. Then, the pellet was resuspended in the 100% acetone, sonicated for 2 min, and covered with aluminum foil and kept at 4°C for overnight. The next day, it was centrifuged for 10 min at 8000 rpm and the supernatant was collected in a fresh test tube and the reading was taken at subsequent wavelength. The supernatant was collected, and its optical density was measured and recorded at 644.8 λ, 661.6 λ, and 470 λ in UV-Visible spectrophotometer (Lichtenthaler, 1987).

The salt-stressed algal cells were withdrawn at day 20 time point, and the algal cells were centrifuged at 8000 rpm for 5 min. Cell pellets were fixed using a fixative containing 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium phosphate (pH 7.0). Scanning electron micrographs were captured using a Scanning Electron microscope operating at 100 kV.

Cells were filtered after harvesting with GC filter paper; the pellet was dried in an oven overnight at 80°C for drying. From that, 1 g of dry algal biomass was processed further for total lipid extraction method. 1 g of dried algal biomass was suspended in 200 ml of chloroform:methanol (2:1), with 500 ml round bottom flask. This setup ran with soxhlet apparatus, for 12 h. Afterward, within the solvent mixture 40 ml of 0.9% saline solution was added, mixed well, and allowed for phase separation. The lipid-containing lower phase was collected in fresh beaker and washed well with methanol and water (1:1) ratio and allowed for phase separation to occur in separating funnel. Lipid-containing lower phase was collected in pre-weighed glass container and kept at room temperature to remove the residual solvent. The lipid yield was quantified gravimetrically, and the same was subjected to TLC or Gas chromatography (Folch et al., 1957; Cristie, 1982).

The total lipid sample was resolved by TLC along with appropriate standards. The transesterification of total lipid was performed as described by Marinkovic and Tomasevic (1998), with little modification. In brief, the cell pellet was re-suspended in 5% of sulfuric acid and methanol (V/V), followed by vigorous vortex for 30 s, and kept in a water bath for 2 h at 90°C, then allowed to cool. 1.5 ml of 0.9% saline solution (w/v) was added to the cooled mixture followed by 1 ml of hexane, vortexed well. The resulting mixture was centrifuged briefly and the upper hexane phase was transferred to a fresh tube. The resulting hexane containing FAME was sequentially washed with sodium bicarbonate followed by water and dissolved using anhydrous sodium sulfate. The dried sample was dissolved in hexane, and fatty acid profile was analyzed by gas chromatography (Su et al., 2011; Xin et al., 2011; Anand and Arumugam, 2015).

The LD mainly comprises a globular neutral lipid core surrounded by a membrane lipid monolayer and it is highly conserved in different species (Murphy et al., 2001). A recent study of LDs in C. reinhardtii showed the abundance of MLDP (Moellering and Benning, 2010; James et al., 2011). The phylogenetic analysis shows that algal MLDP was highly conserved among the aligned sequence of Dunaliella, H. pluvialis, C. reinhardtii, and V. carteri (Davidi et al., 2012). The secondary structure prediction of MLDP shows that they are highly structured α-helices and a short unstructured domain toward the C-terminal end (Guermeur et al., 1998). MLDP comprises a highly conserved motif proline-rich domain of about 21 amino acids in their C-terminal end. All the known MLDPs have two hydrophobic stretch first at 30 and 50 and the second region between 150 and 250 amino acid residues. Even though it is well studied in other green algae, the presence of MLDP, or like proteins was not yet elucidated in S. quadricauda being a suitable strain of biofuel application. The current study confirms the presence of MLDP-like protein conserved in S. quadricauda genome.

Major lipid droplet protein was found out to be the most abundant protein in C. reinhardtii (Moellering and Benning, 2010; Nguyen et al., 2011). Tsai et al. (2014) have reported that MLDP abundance positively correlates with the TAG accumulation, indicating the coregulation of LD and TAG. On the other hand, the lipid MLDP-RNAi silenced lines show about 40% increased LD size in C. reinhardtii under nitrogen-starved condition (Moellering and Benning, 2010). Collectively, in microalgae LD formation, maturation during abiotic stress is tightly regulated through MLDP and its associated partners. Not much information available for S. quadricauda, regarding the presence of MLDP and its abundance during the nitrogen starvation or salt stress condition.

Here, we report that C. reinhardtii MLDP polyclonal antibody (Huang et al., 2013) recognizes and cross-react with the S. quadricauda MLDP, indicating the presence and conservation of MLDP-like protein in green microalga linage (Figure 1). Additionally, we also tested the relative abundance of MLDP in two important stress conditions like nitrogen starvation and salt stress condition; confocal imaging reveals the accumulation of MLDP in both the stress condition (Figure 1). Evolutionally, MLDP or MLDP-like proteins are functionally conserved among green microalgae. However, it is interesting to investigate more about the regulation of MLDP-like protein and its other partners in lipid body maturation during abiotic stress.

The increase in salt concentration exhibits negative effects on the growth of S. quadricauda. The maximum cell density occurs in the control (0.583) (BBM medium having 0.43mM salt concentration) and in 10mM (0.451) salt-treated cells. Increasing concentrations of salt (30, 40, and 50mM) lead to a significant amount of reduction in cell density. A similar effect was also observed in the case of cell number. The maximum cell numbers were recorded at 715 × 10⁴ cells/ml in control, followed by 10 and 20 mM NaCl concentration with 497 and 391 × 10⁴ cells/ml, respectively (Figure 2).

Similarly, Hyder and Greenway (1965) found out that salinity arrests the growth of microalgae. The salt-tolerant studies on C. vulgaris by Talebi et al. (2013) show decrement in growth and moderate resistance up to 0.5M NaCl concentration. Also, salinity more than 0.1M completely inhibited the growth of C. emersoni, and cell density and biomass becomes completely vanished during prolonged stress.

The control S. quadricauda sample had significant SGR compared to higher NaCl-treated sample (0.241–0.134), respectively. An increase (30, 40, 50mM) in the NaCl concentration showed a decline in the SGR and doubling/d (K) occurred in the culture (0.134–0.193), respectively (Table 1). Although the doubling time under salt stress is negatively correlated with the SGR, there was a raise in doubling time (4.98, 5.02, 5.17) in the all higher (30, 40, 50mM) NaCl concentration, respectively. Whereas control had very short doubling time (2.87)(Tt) compared to other higher (30, 40, 50mM) NaCl concentration (Table 1).

The S. quadricauda biomass content was considerably higher at the salinities of control (0.43mM) and 10mM compared to 40 and 50 mM NaCl concentration. The maximum dry biomass content of S. quadricauda was obtained of about 236 mg/L dry biomass in 20th day in control, whereas in 50 mM NaCl concentration the biomass content of 20th day obtained at the 129 mg/L dry biomass (Table 2). The algal biomass production and NaCl concentration are negatively correlated each other in such a way that there was an increment in the biomass production observed at lower NaCl concentrations.

The Chlorella showed the maximum biomass content of about 1.021 g/L under 0.2 M NaCl concentrations. Similar effects were also observed in Chlamydomonas mexicana and Scenedesmus obliquus (Salama et al., 2013; Mohan and Devi, 2014). In contrast, the biomass yield was increased in the salinity stress, i.e., 17–34 mM in green microalgae, Botryococcus braunii, was also reported (Rao et al., 2007).

The maximum lipid yield was observed at 20th day in salt-treated cells compared to control. But the algal growth was slightly inhibited at higher NaCl concentration (40 and 50mM). The total lipid yield was increased in 30, 40, 50 mM NaCl-treated cultures (191 and 205 mg/L) compared to control (118 mg/L) on 15th day (Figure 3). As the trend, the lipid yield was also found to be increased in higher concentration on 20th day. It was about 208, 195, and 201 mg/L in 30, 40, and 50 mM, respectively (Figure 3). It was also further confirmed by TLC with appropriate standard for TAG (Supplementary Image 1).

Similarly, the total lipid content increased under higher salt concentration in Dunaliella were reported by Takagi et al. (2006), and it is possibly due to the cells adapted to the stress condition and accumulation of more lipids. The higher concentration may mediate lipid accumulation by nutrient starvation. And also, the algal cells incubated under saline condition for a longer time period revealed an enhancement in the lipid accumulation within the stressed algal cells. During salt stress condition, pronounced variation in pH, TDS, salinity, and conductivity were observed in Scenedesmus (Supplementary Table 1). According to Rao et al. (2007), the pH of the algal cells varies with respect to salt concentration in green microalgae B. braunii.

The similar pattern of decrease in photosynthetic pigment was also seen in different NaCl concentration. The total photosynthetic pigment level varies gradually viz Chl-a, Chl-b, carotenoids (0.110 ± 0.01, 0.065 ± 0.02, 0.069 ± 0.00 mg/L) in 50 mM NaCl concentration on 20th day (Table 3). The photosynthetic pigments in salt-treated algal cells (50 mM) showed that there is a reduction from 0.377 (Control) to 0.110 g/L of Chl-a on the 20th day of stress. Also, the similar trend was observed with chlorophyll b and carotenoids during salt stress.

There was a report suggesting that the reduction in photosynthetic rate is directly proportional to that of reduction in the growth rate of microalgae (Ben-Amotz et al., 1985). The salt tolerance studies in Chlorella sp. showed that they can tolerate the lower NaCl concentration, and it can grow same as normal cells, but with an increased NaCl concentration revealed degradation of chlorophyll, and it leads to cell death (Salama et al., 2013; Mohan and Devi, 2014). Thus, an optimum NaCl concentration is required for the normal growth of microalgae, higher or lower concentration of salt concentration leads to a decrement in their growth rate (Takagi et al., 2006; Ruangsomboon, 2012).

The fatty acid composition of S. quadricauda under salt stress revealed that there is a marked difference in the saturated: monounsaturated fatty acid composition. The gas chromatogram of control showed that about 79.2% of saturated and 20.8% monounsaturated fatty acids (Figure 4). But salt-treated (50 mM) has about 59.2% of saturated fatty acids, i.e., 20% reduction in saturated fatty acid level. Also, the monounsaturated level is 40.7% which is about 19.9% increment in monounsaturated fatty acids (Figure 4; Supplementary Image 2).

Here, the FAME composition reveals that the altered fatty acid profile is an appropriate proportion for biofuel production. Also, we can mix appropriate proportion of sea water to the media to cultivate the algae in defined concentration, which makes a reduction in biomass production cost and decreases the fresh water requirement for algal cultivation. Abundant availability of seawater can be used for algal cultivation with biofuel application. According to Rao et al. (2007), the presence of monounsaturated and polyunsaturated fatty acids was detected in B. braunii during salt stress with palmitoleic and oleic acids as major fatty acids.

A time course LD formation during nitrogen-starved condition was performed in C. reinhardtii (Moellering and Benning, 2010). The results reveal that first LD was observed at 12 h of N deprivation, followed by the number as well as the large size of LDs formed during 24 or 48 h of N deprivation (Moellering and Benning, 2010). In the present study, we also observed an LD-like structure in scanning electron microscopy shows (Figure 5) more lipid body formation in higher NaCl concentration (50 mM), such structure was not observed in cells grown in normal condition.

The previous reports describe that the Chlamydomonas wild and mutant has accumulated more storage lipid bodies inside the cell by an external environmental factor (Li et al., 2010). In the green microalgae, C. minutissima shows in the oil body formation in environmental stress factors (Wang et al., 2011).