Introduction

Increasing utilization of petroleum-derived fossil fuels is leading to rapid climate changes as a consequence of greenhouse gases (GHGs) emissions [1]. The accelerated exploitation of crude oil reserves makes them unsustainable with the ever-increasing prospect of complete exhaustion. Subsequently, biodiesel from microalgal lipids is projected to act as an alternative, renewable and sustainable source of energy [2]. Microalgae are the most efficient photosynthetic systems having a high growth rate, and can be cultivated in non-arable lands to produce 300 times more lipid than oil crop plants with simple salts as growth requirements [3]. A halotolerant microalga, Dunaliella salina can be cultivated in seawater by naturally avoiding bio-fouling, and being wall-less, the resultant oil extraction is sure to be cost-effective [4]. Wastewater can be utilized to cultivate microalgae that can eliminate up to 90% inorganic nutrients of wastewater simultaneously producing high value biomass at low cost [5]. Due to their high CO2 sequestration capacity, microalgae were also cultivated coupled to industrial flu gas emission in a novel photobioreactor [6]. Adoption of this alternative energy is constrained by the non-availability of a suitable, low-cost photobioreactor for mass cultivation and raising the lipid content in microalgae to a substantial level. The lab has already demonstrated the efficacy of a low-cost, photobioreactor (VFP-PBR) of 32 L capacity for mass cultivation of D. salina and its economic harvesting through FeCl3.6H2O and pH-induced flocculation [7]. The bioreactor yielded 17.85 mgL−1d−1 DCW biomass productivity (BMPr). It is anticipated that increasing the intracellular lipid content up to 60–70% of DCW (dry cell weight) at an appreciable biomass output, may overcome the hurdles of commercial viability [3]. Comparatively higher lipid content was produced in different microalgal species through manipulating culture conditions such as salinity [8], light quality and durations [9], pH [10] or altering levels of the phosphorous [11], nitrogen [10, 11], carbon [12] and sulphur [13] etc. These nutrients, being an integral part of the nucleic acids, proteins, chlorophylls, ATP cycle and signalling processes are capable of directing metabolism of the cell rapidly under suboptimal concentrations, streamlining meagrely available resources for the synthesis of energy and carbon reserves [13]. Nitrogen, a major constituent of the amino acids, is an inevitable requirement in higher amounts for the cell division, growth as well as maintenance of the metabolic synergy [14]. Nitrogen deficiency adversely affected protein synthesis as well as photochemistry of the cells leading to higher lipid accumulation in microalgal species like Dunaliella sp., Ankistrodesmus falcatus KJ671624, Phaeodactylum tricornutum, and Scenedesmus sp. [10,11,12]. Likewise, deficiency of the phosphorous rendered deleterious effects on intracellular signalling, energy balance, ATP turnover, respiration and photosynthesis, ultimately compromising normal cell metabolism as well as growth in microalgal species [13, 14]. However, phosphorous deprivation has also been used as a strategy to enhance lipid accumulation in various microalgal species such as Pavlova lutheri, Isochrysis galbana, and Phaeodactylum tricornutum [15]. Thus, nitrogen and phosphorous limitation has proved efficient in augmenting lipid accumulation in some microalgal species, with compromised biomass productivity. Therefore, lipid-inducing factors such as nitrate or phosphate limitation or depletion coupled with stress alleviating supplements such as micronutrients [16] and bicarbonate [12] were optimized for overproduction of lipids vis-à-vis biomass. Simultaneous manipulation of Fe, Na and N levels in the cultures of C. minutissima, yielded higher lipid over that of individual nutrient treatment [17]. Again, C. vulgaris cultures subjected to simultaneous nitrate limitation (0.03 mM) and NaCl stress (0.11 M) resulted in higher lipid productivity (160 mg L−1 DCW) [18]. Microalgae assimilate carbon either in the form of inorganic carbon (HCO3 −) or gaseous CO2 [12]. Saline microalgae such as D. salina, usually grow under higher pH, where lower solubility of CO2 renders gaseous CO2 unavailable to the growing cells. Such situation demands supplementation of the medium with NaHCO3 which is cheaper, easy to handle, and effective in small amounts, thereby, significantly nullifying inorganic carbon unavailability [13]. D. salina also possesses external and internal carbonic anhydrases, enabling it to efficiently utilize the bicarbonate ion and maintain photosynthesis in hypersaline condition [19, 20]. In microalgae, acetyl CoA carboxylase (ACCase) serves as the biocatalyst in the first and rate-limiting step of the lipid biosynthesis, converting acetyl-CoA to malonyl-CoA. Beta carboxyl transferase (β-CT) is known to be the key sub-unit of heteromeric ACCase of chloroplasts, being encoded by the plastidic accD gene [21]. It was reported that lower expression of accD brought down the activity of ACCase and vice versa in tobacco plant [22]. Thus, understanding accD expression pattern in the target organism under varied nutrient regimes may facilitate genetic engineering prospects after maximizing the lipid content through nutrient manipulation strategies. Likewise, RuBisCO, the enzyme involved in the fixation of CO2 via the Calvin-Benson cycle is coded by rbcL (large sub-unit) and rbcS (small sub-unit) and thus, central to the biomass production [23]. Therefore, it was imperative to monitor the expression of rbcL and rbcS genes. There are limited studies with a focus on the inter-relationship of nutrient conditions, photosynthetic performance, and key gene expression patterns in D. salina [15, 24].

Nitrogen limitation strategies to overproduce lipids in microalgae are mainly two types; (a) two-stage cultivation involving culture of microalgal cells in nutrient sufficient medium in the first stage and then transferring to nitrogen deficient medium in second stage [18, 25], (b) one-stage cultivation with the addition of desired nitrogen only and make the cells starve for nitrogen in the due course which may be advantageous over two stage considering the cost and time consumption [26]. Nevertheless, the biomass and lipid accumulation in microalgal system in response to varied nutrient levels is species-specific [27]. Therefore, in the present work, D. salina was investigated for higher lipid production in one stage without compromising the biomass output. The biomass may be used for co-production of commercially valuable compounds such as lipids, glycerol, carotenoids and therapeutic compounds that can offset high cost involved in production [25]. The target strain D. salina was collected from a hypersaline lake, Sambhar, from Rajasthan, India. This strain showed tolerance in range of NaCl from 0.25 to 5.0 M [20]. The strain grew optimally at 0.5 M NaCl in the batch culture. It is a cell factory for many high value compounds, a natural β-carotene source, having high intrinsic lipid production capability and immense environmental stress resilience making it suitable for cultivation under hot climate without requiring fresh water [28]. There are also reports that explored the ways to increase the biomass and lipid production in halophilic D. salina by varying the levels of different nutrients and other factors [29]. Most of these studies lacked the strategy involving the determination of threshold limits of different nutrients for simultaneous improvement of biomass and lipid production and also a better understanding of the physiological status of the cells. A multiphasic approach involving nutrient manipulation, assessment of its impact on photosynthetic performance and expression of key genes involved in photosynthesis and lipid production of D. salina might be useful in developing a suitable way for hyper-accumulation of lipids while maintaining biomass productivity, and thereby paving the way in reducing the cost of biofuel production, as the biomass produced may also be used as a source of valuable products and feedstock for biofuel. Therefore, the objective of the present study was to determine the suitable concentrations of different nutrients (N, P and C) for maximizing lipid yield from D. salina while maintaining optimum biomass productivity in one-stage cultivation. Also, their effects on biomass and lipid production were correlated with the photosynthetic performance of the cells as well as expression of key genes, accD (involved in lipid biosynthesis), rbcL and rbcS (involved in inorganic carbon assimilation and biomass production).

Materials and Methods

Organism and Culture Conditions

Modified Johnson’s Medium (MJM) was used to prepare high-density cultures of D. salina for inoculating the treatments [5]. The basal MJM contained 7.38 mM MgCl2.6H2O, 2.03 mM MgSO4.7H2O, 2.68 mM KCl, 1.36 mM CaCl2.2H2O, 10.00 mM KNO3, 0.5 mM NaHCO3, 0.25 mM KH2PO4, 2.81 µM Na2EDTA, 4.5 µM FeCl3.6H2O, 0.98 µM H3BO3, 0.03 µM (NH4)6Mo7O24.4H2O, 0.024 µM CuSO4.5H2O, 0.021 µM CoCl2.6H2O, 0.03 µM ZnCl2, 0.018 µM MnCl2.4H2O. Media were supplied with 0.5 M NaCl at 80 µmolm−2 s−1 illuminations, light/dark photoperiod of 16/8 h and maintained at 28 ± 2 °C. Sterile media and glass-wares were used under a laminar flow hood (Instech, India) to carry out all operations. All chemicals used were of analytical grade (Merck, India).

Experimental Design

The current experiment involved three steps. In step 1, different concentrations of KNO3 (0.00, 1.25, 2.50, 5.00 and 10.00 mM) and KH2PO4 (0.00, 0.064, 0.128, 0.256 and 0.512 mM) were employed one at a time to assess the impact of nutrient stress on the growth, photosynthetic efficiency, and lipid production in D. salina. During step-1 of the experiment, the concentration of KH2PO4 was kept at 0.256 mM (basal level) with variation of KNO3 levels. Conversely, under different KH2PO4 level, 10.00 mM KNO3 was used. Each experiment continued for 12 days and conducted in triplicates of biological samples (total 27 treatments) and the data recorded in the form of mean ± SD. A combination of nitrate (‘Nlim’ = 1.25 mM KNO3, 1/8 of the basal level and ‘–N’, 0.00 mM KNO3) and phosphate (-P = 0.000 mM KH2PO4 and Plim = 0.064 mM KH2PO4, 1/4 of the basal) that resulted in higher lipid production in step 1 were taken for further study in step 2 and evaluated in triplicates for the above parameters (total 15 experiments). Step 3 involved the addition of 5 (C1) and 20-fold (C2) NaHCO3 over that of basal medium (C1 = 2.5 mM, and C2 = 10.00 mM) to the combined nutrient stress of Step 2 and examined for overall growth, biomass productivity, and lipid yield (total 27 experiments). The photochemistry of such cells along with relative expression of RuBisCO (carbon assimilation) and accD (lipid biosynthesis) genes were also examined. In all the cases, MJM containing 10 mM KNO3, 0.5 mM NaHCO3, and 0.256 mM KH2PO4 (basal level of respective nutrients) was taken as control. The experimental design is briefly described in the following flow chart (Fig. 1).

Fig. 1
figure 1

Schematic representation of the experimental design of the current work

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Estimation of Biomass Productivity

For estimating biomass productivity, the cultures were filtered through 0.45 µm Whatmann glass filter paper (Sartorious, Göttingen, Germany) and the resulting pellet was oven-dried at 80 °C for 12 h. Biomass productivity (BMPr, mgL−1d−1 dry cell weight, DCW) was measured using the following formula [25];

$$\mathrm{BMPr}=\left({\mathrm{BM}}_{2}-{\mathrm{BM}}_{1}\right)/\left({\mathrm{T}}_{2}-{\mathrm{T}}_{1}\right)$$

Here, BM1 and BM2corresponded to dry cell weight of cultures at time T1 and T2 respectively measured in an analytical balance (Sigma Aldrich, USA).

Photopigment Estimation

For determining the pigment contents, microalgal culture (2 mL) was centrifuged (Eppendorf 5415R, Hamburg, Germany) at 10,000 rpm, 4 min at room temperature and the pellet was resuspended in an equal volume of 99.9% methanol and incubated at dark (24 h at 4◦C). Pigment concentrations were determined after taking absorbance at 470, 652.4, and 665.2 nm in a UV–VIS spectrophotometer (Thermo Scientific Evolution 160, v8.01, Massachusetts, USA). The following formula was used for pigment content estimation [30].

$$\begin{array}{lll}\mathrm{Chl}\;a\left(\mathrm{{\mu}g}/\mathrm{mL}\right)=16.72{\mathrm A}_{665.2}-9.16{\mathrm A}_{652.4}\\\mathrm{Chl}\;b\left(\mathrm{{\mu}g}/\mathrm{mL}\right)=34.09{\mathrm A}_{652.4}-15.28{\mathrm A}_{665.2}\\\mathrm{Carotenoids}\;\left(\mathrm{{\mu}g}/\mathrm{mL}\right)=\left(1000{\mathrm A}_{470}-1.63\;\mathrm{Chl}\;a-104.9\;\mathrm{Chl}-\mathrm b\right)/221 \end{array}$$

Lipid Content Estimation

Total lipid was extracted and quantified gravimetrically following the method of Bligh and Dyer [31]. Briefly, the culture of appropriate density was harvested (4000 rpm × 4 min, Remi R-8C, Mumbai, India) and washed twice with double-distilled water. The algal biomass was vacuum freeze-dried (Martin Christ, Alpha 1–2 LDplus, Germany), 100 mg of the dried biomass was taken in a 10 mL falcon tube (Tarson, India) to which 3 mL methanol + chloroform (2:1) was added. Then, the tube was kept at 25 °C for 24 h. Then, it was vortexed and sonicated for 10 min at 20 kHz (VCX 500, Sonics & Materials Inc, USA) with intermittent shaking. To it, 1 mL chloroform was added and mixed vigorously. This was followed by addition of 1.8 mL saturated NaCl solution and vortexing the mixture for 2 min. The mixture was then centrifuged for 15 min (3000 rpm, Remi R-8C, Mumbai, India) and bottom organic phase was transferred to a previously weighed clean vial (V1). The organic bottom phase was evaporated in the thermo block (95 °C) under nitrogen stream. In order to completely remove the solvent, the residue was further dried (104 °C × 30 min). Now, the weight of the vial was again monitored (V2) and the lipid content was calculated as, V2 – V1 and expressed in terms of mg g−1 of dry cell weight.

Neutral Lipid Analysis

To detect the accumulation of neutral lipids, D. salina cells were stained with Nile red (9-diethyl amino 5-hexabenzo α-phenoxazine-5-one, Sigma, USA) soon after washing thrice with fresh media. The quantitative determination of neutral lipids was done by exciting the NR stained cells at 485 and detecting the emission at 612 nm using a fluorescence spectrophotometer (Cary Eclipse, Agilent, USA) following the procedure described by [32]. NR-stained D. salina micrographs with prominent golden yellow emissions (lipid bodies) were imaged using Zeiss LSM 780 laser-scanning confocal microscope–equipped Zen 2010 imaging software (Carl Zeiss, GmbH, Germany) with a common exposure time [33]. The excitation and emission wavelengths were at 525 and 595 nm respectively.

Pulse Amplitude Modulated (PAM) Fluorometry

Photosynthetic performance of the cells under various nutrient treatments was measured with reference to maximum photochemical quantum yield (Fv/Fm) of PS II using PAM Fluorimeter (PAM-2500; Heinz Walz, Effeltrich, Germany). Rapid Light Curve (RLC) generation and data processing were done using PAM-2500 software (v 3.20) [20]. Before the measurement, liquid samples were placed in the dark (20 min) for closing all photosystems. Chlorophyll fluorescence at 0.001 and 3000 µmolm−2 s−1 light intensities were taken for obtaining Minimal (Fo) and maximum fluorescence (Fm) levels. Fv/Fm, representing the maximum quantum output of PS II was calculated using the following formula [34];

\(\frac{Fv}{Fm}=\frac{Fo-Fm}{Fm}\), where, Fv is the differential fluorescence value.

Calculation of relative electron transfer rate (rETR) was done as per the formula described in [34]; rETR = PAR · ETR-Factor · Y(II) · PPS2/PPPS, where ETR-Factor (default value was 0.84) depicts the fraction of incident light absorbed by photo-pigments; Y(II) corresponds to effective quantum yield of PS II; PPS2/PPPS: ratio of photons absorbed by PS II and all photo-pigments (default value was 0.5 assuming uniform electron transfer rates across PS I and II). The highest value for rETR across 0 to 2000 μmol photons m−2 s−1 PAR range was designated as rETRmax.

Calculation of Non-photochemical quenching (NPQ) was done as per the formula described by [34];

$$NPQ=\frac{Fm-Fm\mathrm{^{\prime}}}{Fm\mathrm{^{\prime}}}$$

Here, Fm' stands for fluorescence maxima during a pulse of non-saturating light.

RNA Extraction, RT-PCR, and Gene Expression Analysis

For total RNA extraction, reverse transcriptase PCR (RT-PCR) and expression profiling of different genes, the methods used were described by [35]. For RNA extraction, the aliquot of cultures (2 mL, OD750 nm = 0.6) was sampled at 0, 6, and 12th day of cultivation. The biomass pellet was collected at 2000 rpm, 5 min (Eppendorf 5415R, Hamburg, Germany), and washed with DEPC water before grounding it with mortar and pestle under liquid nitrogen. Extraction of total RNA was performed using QIAGEN RNAeasy Mini Kit (Qiagen, Germantown, USA) as per the supplemented instruction. Agarose gel (1.2%) electrophoresis and spectro-photometric analysis (NanoDrop One, Thermo Scientific, Massachusetts, USA) were done to ensure the quality of extracted RNA. Subsequently, High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems™, Massachusetts, USA) was used to synthesize the first strand of cDNA from total RNA adopting the manufacture’s protocol. The cDNA obtained from this acted as a template for semi-quantitative RT-PCR assay. Reverse transcription was run in a thermocycler (BioRad T100 Thermal Cycler, Hercules, USA) Primers targeting important genes of lipid synthesis (accD) and carbon fixation (rbcL and rbcS) were designed using the NCBI Blast tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome), synthesized (Agrigenome Labs, Kochi, India), and used for quantitative detection of expression of these genes (Table 1). PCR was performed with a final volume of 25 µL and the reaction mixture contained 25 ng of template cDNA, 200 µM dNTPs, 10 pM each of forward and reverse primers, 25 µM CaCl2, 10X reaction buffer (2.5 µL), and Taq DNA polymerase (0.25 U, Takara, Japan). The reaction condition was 1 cycle of 94 °C (3 min); 25 cycles of denaturation at 94 °C (30 s), annealing at 51/48.8/48.5 °C (30 s) and extension at 72 °C (30 s); final extension for 5 min (72 °C). Sterile DEPC (Diethyl PyroCarbonate) treated Milli-Q® (Millipore, USA) water was used to inactivate RNAse enzyme in water and lab utensils. RNAse-treated Milli-Q water was taken in place of cDNA template to act as a negative control. D. salina actin (D-actin) was taken as reference for the purpose owing to its consistent level of expression across the treatments [36]. The template for designing accD primers was taken from complete CDS of Dunaliella salina acetyl CoA carboxylase beta subunit (GenBank: EF363909.1). The templates for rbcL and rbcS primers were obtained from conserved sequences of respective genes after aligning the mRNA sequences chosen from eight related microalgal species of D. salina. All reactions were performed in triplicates and the products were run in 1.2% agarose gel. Gel Doc 2000 system provided with volumetric analysis tool was used to quantify the signal generated in gel (BioRad, Hercules, USA).

Table 1 Primer sequences for semi-quantitative expression analysis of accD, rbcL and rbcS genes
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Statistical Analysis

Experiments were operated in triplicates and the results were presented as mean ± SD. One-way Analysis of Variance (ANOVA) was used to validate the statistical significance of the treatments adopting Tukey’s multiple range test at α = 0.05 (95% confidence level) provided in the SPSS statistical package (SPSS Inc Version 20.0, IBM). The value p < 0.05, denotes the statistically significant difference between the obtained values of respective parameters.

Results and Discussion

Effects of Nitrate or Phosphate Limitation

Nitrogen and phosphorous are vital macronutrients for the cell division, growth, and smooth functioning of the cellular machinery, thus affecting biomass and lipid production. Photoautotrophic growth and biomass productivity (BMPr) in D. salina at 0.00 –10.00 mM KNO3, showed concentration-dependent response (Fig. 2A). Such responses have also been reported in other microalgal species [11, 13]. The highest BMPr (25.25 ± 0.86 mgL−1d−1) was recorded at the basal level of nitrate (10.00 mM) and lowest (11.05 ± 0.59 mgL−1d−1) in nitrogen-deficient cultures of D. salina (Table 2). BMPr decreased with the reduction in the nitrate concentration of the culture, indicating that more than 2.50 mM nitrate was required for higher biomass productivity. A minimal requirement of nitrate (1.25 mM, 1/8 of the basal level, called as ‘Nlim’) seemed to be essential for biomass productivity (20.04 ± 0.26 mgL−1d−1). However, there was increase in BMPr with increasing KH2PO4 concentration up to the basal level of the phosphate (0.256 mM), but decreased sharply by 0.34-fold (16.98 ± 2.09 mgL−1d−1), when phosphate level was increased to 0.512 mM (Fig. 2B and Table 2). This decrease in BMPr was very close to –P cultures (0.40-fold).

Fig. 2
figure 2

Photoautotrophic growth behaviour of D. salina under varying concentrations of (A) KNO3 (0.00 –10.00 mM), (B) KH2PO4 (0.00 – 0.512 mM), and (C) concomitant nitrate and phosphate limitation (-N and Nlim referred to 0.00 and 1.25 mM KNO3 while –P and Plim referred to 0.00 and 0.064 KH2PO4 respectively). Each value is represented as mean ± SD of three replicates

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Table 2 Quantification of biomass productivity (BMPr), lipid content (LC), photosynthetic quantum yield (Fv/Fm), total chlorophyll (TChl) and carotenoids under different doses of nitrate and phosphate. Different letters in the superscript denoted the significant values (p < 0.05) among different nutrient levels
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Interestingly, when –N and –P cultures were compared, –P cultures yielded 38% higher biomass (15.31 ± 0.38 mgL−1d−1) over that of –N cultures (11.05 ± 0.59 mgL−1d−1). This difference in BMPr was validated with the difference in photosynthetic quantum yield under –P cultures (Fv/Fm = 0.558 ± 0.08) and –N cultures (Fv/Fm = 0.396 ± 0.11) (Table 2). Relatively better growth in –P cultures over –N cultures might be due to the supply of the phosphate from intracellularly stored polyphosphate bodies [37, 38]. The data (Ref. Table 2) indicated that even 1/4 of the basal level of phosphate (0.064 mM, called ‘Plim’) was sufficient to sustain the growth. This data is supported by observations of [24], who have shown a relatively modest decrease in growth under phosphate deprivation over that of nitrate in the marine microalgal species. Phosphorus has been demonstrated to have hormesis effect on the viability of another green alga, Chlorella regularis cells under nitrogen limitations [38]. The excess of phosphate inhibited the growth of C. regularis by formation of excess of polyphosphate bodies and thereafter binding with the proteins, resulting in protonated amide nitrogen [38]. The latter damages the membrane, rendering disorganization of cellular organelles and deformation of the cell wall [38].

Total chlorophyll (TChl) content of D. salina cells reduced marginally (4.1%) in 1/4 of the basal nitrate (2.50 mM) compared to control. However, further decrease in nitrate level (–N or Nlim condition) reduced chlorophyll content, lowering photosynthetic quantum yield, consequently lowering BMPr (Ref. Table 2). This result was consistent with the other reports showing that nitrogen deficiency in D. salina and other microalgae led to drastic reduction in photosynthetic efficiency, thereby reducing the BMPr [13, 24]. The substantial reduction in TChl was coupled with increase in carotenoid content which might have acted as shock absorber due to quenching of reactive oxygen species, ROS [39].

The Pulse Amplitude Modulator technique which measures Chl a fluorescence was used to evaluate the physiological status and the photosynthetic efficiency of microalgal cultures [40]. It was demonstrated that the microalgal cultures under stress can be described in terms of chlorophyll fluorescence when Fv/Fm values measured below 0.4 [41, 42]. Photosynthetic quantum yield recorded was significantly higher (p < 0.05) in the cultures with 1.25 mM KNO3 compared to nitrate-deficient cultures, signifying the importance of nitrogen in maintaining photosynthetic activities even under sub-optimal availability (Table 2). Sustained photosynthetic efficiency (Fv/Fm ≥ 0.558) in –P and Plim cultures may be due to existing intracellular reserve of phosphate. Contrasting to nitrate, a very low amount of phosphate was sufficient for the growth of D. salina cells. The presence of nitrogen in the metabolic hub was of paramount importance as it changes the rate of cell metabolism significantly [14, 27]. Highest LC (298.24 ± 19.9 mg g−1 DCW) was recorded at 1.25 mM nitrate (Nlim), therefore, it may be considered as the critical threshold limit for higher lipid accumulation in D. salina. Similar reports in other microalgae suggested that reduction of nitrate level to half of the ambient (17.65 mM) resulted in higher lipid production (313.12 mg g−1 DCW) in Ankistrodesmus falcatus KJ671624 [11], and diatoms also produced higher lipid under limited nitrogen over deficient ones [12, 43]. Likewise, maximum LC (264.5 ± 14.0 mg g−1) was recorded in –P condition among different phosphate concentrations. Phosphate depletion also induced higher lipid accumulation in D. salina [13] and Chlorella sp. [44]. In another report, phosphate starvation in f/2 media resulted in 290 mg g−1 DCW lipid production in D. salina cells [24]. Thus, assuming that simultaneous application of nitrate and phosphate deficiency could further accelerate the lipid accumulation in D. salina cells, combinations of nitrate and phosphate levels were selected taking 1.25 or 0.00 mM nitrate and 0.00 or 0.064 mM phosphate as these concentrations yielded maximum lipid among all the culture conditions. This sort of strategy with combined nutrient deficiency has been employed in several other microalgal species with considerable success [10, 45].

Effect of Combined Nitrate and Phosphate Limitations

Photoautotrophic growth of D. salina under selected combinations of nitrate and phosphate is shown (Fig. 2C and Table 3). The upper curve demonstrated that the control culture grew better (BMPr = 23.24 ± 2.78 mgL−1d−1) compared to others.

Table 3 Effect of concomitant nitrate and phosphate limitations on BMPr, lipid content, photopigments and photosynthetic quantum yield of D. salina cultures. Different letters in the superscript denoted the significant values (p < 0.05) among different nutrient levels
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The combined deficiency of N and P (-N-P) had a more deleterious impact on the growth of D. salina than that of the individual nutrient depletion in the culture medium. A similar trend was reported by [46] in a different strain of D. salina under –N-P condition, although only 228.5 mg g−1 DCW lipid could be accumulated. In the present study, a new strategy was adopted through addition of low level of nitrate (Nlim) or phosphate (Plim) to –N–P cultures, which ameliorated the stress considerably, as reflected by an increase in the BMPr (Ref. Table 3). However, BMPr recorded in NlimPlim cultures was significantly higher (p < 0.05) over the other combinations, 16.14% lower than the control. Highest Fv/Fm (0.603 ± 0.18), TChl (55.07 ± 4.85 mg g−1 DCW) were recorded in the control cultures having carotenoids at the lowest level (12.19 ± 1.52 mg g−1 DCW). NlimPlim cultures had slightly lower Fv/Fm (13%), TChl (2% lower), but increased carotenoid content (15%) over that of the control cultures. This indicated that addition of low levels of N and P helped in the sustenance of the algal growth.

Lipid content enhanced significantly (p < 0.05) in all the combinations when compared to the control or respective individual nutrient-limited conditions (Table 3). Highest LC (341.16 ± 27.5 mg g−1 DCW) was recorded in Nlim–P followed by that of NlimPlim cultures (319.22 ± 24.0 mg g−1). Relatively modest BMPr in Nlim-P (13.12 ± 0.57 mgL−1d−1), indicated the scope of BMPr improvement. Phosphate deprivation may have triggered the early onset of lipid accumulation in Nlim–P cultures, and therefore, the cells were able to channelize the resources towards lipid production using minimally available nitrate. In another report of algal species (C. sorokiniana), reducing nitrate to half (5.88 mM) and phosphate to one-fourth (0.057 mM) of the ambient BG-11, resulted in rise in lipid content from 185 to 330 mg g−1 DCW [16]. Simultaneous nitrate limitation and phosphate starvation also yielded 2.55-fold more lipids in A. falcatus KJ671624 cells as compared to the control [10]. Therefore, BMPr was enhanced to a substantial level while producing comparatively higher lipid, employing NaHCO3 augmentation strategy in the subsequent part of the study.

Effects of NaHCO3 Augmentation in Nutrient Limitation Conditions

Determination of Critical Threshold Level of NaHCO3

The possibility of enhancing biomass productivity while keeping higher lipid production in the microalgae can be accompanied through increasing the carbon flux in the organism by supplying more inorganic carbon. The solubility of the atmospheric CO2 significantly decreases in the marine and hyper-saline conditions. Thus, inorganic carbon (IC) plays a limiting factor in these culture systems, affecting photosynthesis and triggering enhanced carbon concentrating mechanisms (CCM) in microalgae [47]. Bicarbonate in the medium acts as a buffer (pKa = 10.3) and helps in supplying nutrition to the microalgal cell in the form of inorganic carbon [48]. Addition of NaHCO3 in D. salina cultures also proved to be effective in enhancing the relative percentage of saturated and mono-unsaturated fatty acids that are suitable for biodiesel production [7]. Therefore, to determine critical concentration of NaHCO3 for enhancing biomass and lipid production under combined nutrient limitations, the D. salina cultures were augmented with varying concentrations of NaHCO3 (0.50 to 20.00 mM) (Fig S1, supplementary information). The increase in BMPr of such D. salina cultures up to addition of 10.00 mM bicarbonate may be attributed to the enhanced availability of the carbon skeleton, higher TChl content leading to robust photosynthetic quantum yield (Fv/Fm > 0.6) with lowered carotenoid content, indicating the role of bicarbonate in alleviation of the nutrient (N or P) stress (Table S1, supplementary information). The addition of bicarbonate beyond 10.00 mM in the present experiment was non-conducive for algal growth as it might have drastically changed the pH of the media, causing salt formation. Thus, the highest BMPr was obtained at 10.00 mM NaHCO3 supplemented D. salina culture, although lipid content in these cultures (0.50 to 10.00 mM) did not vary significantly (Ref. Table S1). Therefore, it was aimed to evaluate 2.50 and 10.00 mM NaHCO3 augmented D. salina cells growing in individual pr combined nutrient (N and P) deficiency or limited condition for enhancing their BMPr as well as lipid yield.

NaHCO3 Addition to Individual Nutrient-limited Cultures

D. salina grows at high pH, therefore, supplementation with higher level of NaHCO3 in culture medium may overcome the lower availability of the dissolved inorganic carbon in media under high pH, resulting in better biomass as well as lipid productivity in this and other microalgal species [12, 13, 49,50,51]. The photoautotrophic growth of D. salina in the media, having individual nutrient deficiency (-N or –P) or limitation (Nlim or Plim), upon augmentation with 2.50 or 10.00 mM NaHCO3, showed the highest BMPr (57.03% more than control) in Nlim + C2 (1.25 mM nitrate + 10.00 mM NaHCO3) cultures (Fig. S2 and Table S2, supplementary information). Similar findings were reported in the diatom RGd-1 and the green alga Scenedesmus sp., where cultures amended with high NaHCO3 resulted in faster growth [48, 52]. In terms of lipid accumulation, nitrate starved cultures resulted in increased cellular lipid content upon the addition of NaHCO3 (Fig S3, supplementary information). It was also demonstrated in another green alga Scenedesmus sp. that increasing NaHCO3 level under nutrient starvation conditions led to de novo synthesis of lipids, and the carbon flux is channelized towards lipid anabolism [52]. Again, the role of HCO3 − was highlighted in augmenting the PS II electron transport efficiency as well as assembly of the Mn4CaO5 inorganic core of the oxygen evolving complex, thus validating the consensus that IC in the form bicarbonate can not only serve to replenish depleting CO2 in the medium under high pH, but also ameliorate cellular stress caused by possible factor [53]. However, Chavoshi and Shariati have reported that D. bardawil accumulated around 400 mg g−1 DCW of lipid upon treatment with 100 mM acetate (organic carbon), keeping the KNO3 concentration at 5.00 mM, suggesting that the mixotrophic growth may be useful for higher lipid production [54]. This indicates that the carbon flux increases either in the form of inorganic or organic carbon supplied to microalgal cells, inducing higher lipid production. There is also a report of increased lipid content (up to 540 mg g−1 DCW) in D. salina using De Walne’s media augmented with 100 mM NaHCO3, [13]. In contrast, in the current study, NaHCO3 levels beyond 20 mM formed precipitation (in MJM media), thus, limiting its application at a very high concentration. Therefore, the quantitative application of inorganic carbon seemed to be species-specific, and it largely depends on the medium composition.

NaHCO3 Addition to Combined Nutrient-limited Cultures

Effect of NaHCO3 addition on the photoautotrophic growth, biomass, and lipid production of D. salina cells under simultaneous nitrate and phosphate limitations is shown in Figs. 3A-E. The data revealed that the addition of bicarbonate to combined nutrient stress cultures whether limited or deficient for N and P, increased the BMPr. This again emphasized that carbon supplementation played important role in biomass production. The biomass productivity of D. salina cells was much dependent on the presence of nitrogen over phosphorus, although the latter was also necessary for the sustenance of the alga. Phosphate deficiency in combination with 1.25 mM KNO3 (Nlim-P) resulted in lower biomass (13.12 mgL−1d−1 DCW). The addition of 10.00 mM NaHCO3 to such cultures significantly enhanced the BMPr to 1.27 and 2.25-fold over the control and Nlim-P cultures respectively. Thus, augmented NaHCO3 might have enabled the D. salina cells to overcome low availability of CO2 in high pH media. As far as lipid content was concerned, a substantial increase was recorded in Nlim-P cultures supplemented with 10.00 mM NaHCO3 reaching 400.1 ± 25.1 mg g−1 DCW which was 2.02 and 1.17-fold over that of the control and Nlim-P cultures without 10.00 mM NaHCO3 respectively. This increment in BMPr compared to Nlim –P cultures might be affiliated to the stress ameliorating effect of bicarbonate which helped in maintaining the photosynthetic health of the cells as reflected by robust photosynthetic performance. The enhancement in lipid content may be correlated to the stress triggered out of phosphate deficiency at the beginning of the culture. Addition of higher bicarbonate only provided more carbon skeleton and energy (ATP) through robust photosynthesis for still higher lipid production [12, 13]. The lipid content in bicarbonate-augmented nitrate and phosphate-deficient cultures was in consonance with the reports for other microalgae such as Scenedesmus sp. [52], P. lutheri [55], and Chlorella sp. [56]. Bicarbonate-amended media (10.00 mM) under nitrate deficient conditions, showed relatively lower improvement in the BMPr owing to lowered chlorophyll content (< 48.66 ± 4.18 mg g−1 DCW) and photosynthetic inefficiency (Fv/Fm < 0.395 ± 0.17). Ameliorating effects of bicarbonate under nutrient stress has also been reported that showed increase in antioxidant activities of another microalga, Pachycladella codatii, thus playing a role in neutralizing ROS [57]. Gordillo and co-workers had also established the decreased carbon fixing ability in S. platensis under nitrate deprivation, despite the fact that inorganic carbon was available in the plenty [58]. It was inferred from the data presented in Fig. 3 that Nlim-P + C2 or NlimPlim + C2 cultures were appropriate for the biomass vis-à-vis lipid production.

Fig. 3
figure 3

Effect of NaHCO3 augmentation on photoautotrophic growth behaviour of D. salina cells under (A) both nitrate and phosphate deprivation (-N –P), (B) nitrate deprived and phosphate limitation (-N Plim), (C) nitrate limited and phosphate deprivation (Nlim –P), and (D) both nitrate and phosphate limited condition (NlimPlim) and Fig. 3E showed the effect of NaHCO3 augmentation on BMPr and LC in D. salina cultures after 12 d under concomitant KNO3 and KH2PO4 limitation. C1 ad C2 referred to addition of 2.50 and 10.00 mM NaHCO3 to the respective medium. Values are represented as mean ± SD with triplicate readings. Different letters suggest to significant difference (Tukey’s test; p < 0.05) in BMPr or lipid content at different nutrient conditions

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Analysis of Effects of Individual/Concomitant Nutrient Limitations and NaHCO3 Addition at Different Time Points

It was clear from the preceding results that among the nitrate, phosphate, and bicarbonate treatments, nitrate was the most important nutrient for biomass as well as lipid production. It also helped in maintaining the cells healthy and even photosynthetic performance of the algal cultures. To understand the temporal role of each factor, and change in relative carbon flux towards lipid biosynthesis, a comparative analysis was done at three-time points i.e., 0, 6, and 12th d of the selected cultures. The experimental data including gene expression profiling was done at 0th d after 2 h of incubation period to allow stabilization of the cultures. Cultures at day 6 and 12 represented the logarithmic and stationary phases of the experiments respectively.

Effects on Biomass Productivity and Neutral Lipid Accumulation

Temporal variations in the BMPr, neutral lipid accumulation, and photosynthetic performance of D. salina cultures grown at different nutrient regimes are shown in Figs. 4 and 5. It was already seen that 10 mM NaHCO3 (C2) addition in all the cultures proved better for both biomass and lipid productions, and therefore, only C2-augmented cultures were selected for comparative analysis keeping aside C1-augmented cultures. The significant decrease in BMPr in Nlim-P + C2 and NlimPlim cultures after day 6 might be due to consumption of limited nitrate content (1.25 mM) that could again be the reason why neutral lipid content increased sharply in these cultures after the 6th d. Consequently, the production of neutral lipid was enhanced significantly both at 6th and 12th d under nutrient deficiency conditions. Higher values of NRF (nile red fluorescence) in –N and Nlim–P cultures at 6th d over that of Nlim (p < 0.05) suggested the immediate induction of lipid biosynthesis owing to the absence of nitrate and phosphate in these cultures respectively. Neutral lipid level reached an equal level in the Nlim and NlimPlim cultures at the 12th d which can be ascribed to exhaustion of limited nitrate available in the media. From this data, it can also be inferred that limited phosphate may have been sufficient for growth and did not cause significant stress. The increase in the neutral lipid content was qualitatively verified with confocal fluorescence micrographs of Nile Red stained D. salina cells at the 12th d showing prominent golden yellow fluorescence in –N, Nlim, Nlim–P and Nlim –P + C2 culture cells (Fig. S4, supplementary information).

Fig. 4
figure 4

Variation of BMPr and LC in different nutrient limitations along with NaHCO3 augmentation at three different time points; (A) biomass productivity and (B) neutral lipid accumulation in terms of Nile Red fluorescence. The mean value ± SD of triplicate readings are represented as vertical bars. Different letters suggest that means are significantly different (Tukey’s test; p < 0.05) with upper case letters stand for significant differences in folds of biomass productivity or Nile Red fluorescence at various time points within the same nutrient treatment while lower case letters signify significant differences in parameters between different nutrient conditions at same time points

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Fig. 5
figure 5

Photosynthetic performance of D. salina cells exposed to deficiency or limitation of nitrate, phosphate individually or in combination or augmented with 10.00 mM NaHCO3 (C2); (A) Maximum photosynthetic quantum yield (Fv/Fm), (B) relative electron transport rate (rETR), and (C) non-photochemical quenching (NPQ). Values represented are mean ± SD of triplicate readings

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Photosynthetic Status of D. salina Cells under Different Nutrient Conditions

The maximum quantum yield of cells at the 6th d was most adversely affected in –N (0.42 ± 0.02) and –N + C2 (0.55 ± 0.03) which reduced to 0.17 ± 0.01 and 0.19 ± 0.027 respectively at the 12th d, suggesting malfunctioning of photosystems due to nitrate non-availability (Fig. 5). Gao and co-workers had also recorded a sharp decline in Fv/Fm (17%) and rETR (36%) upon nitrate deprivation in D. salina [24]. Among the nitrate limited conditions without extra bicarbonate, Fv/Fm value was lowest in the Nlim–P followed by that of Nlim and NlimPlim. Thus, the absence of phosphate under Nlim condition had greater detrimental effect on photosystems over phosphate availability. However, Nlim and Nlim–P showed similar quantum yield at the 12th d signifying the exhaustion of nitrate at the end of the culture period in both the sets. Similarly, the performance of photosystems was completely compromised of –N and –N + C2 cells at the 12th d, inferring that extra bicarbonate can only have a limited ameliorating effect under nitrate-deprived condition, hence, improved quantum efficiency in all the Nlim cultures. Interestingly, rETR values in NlimPlim condition (28.97) were superior to control (25.96) and NlimPlim + C2 (28.76) owing to the cell’s desperate measure to sustain under such nutrient limitation with the availability of nitrate and phosphate at a critical level to maintain cellular photochemistry. The surplus light energy is dissipated in the form of heat (NPQ), keeping the entropy of photosystems under check. Data revealed the rise in NPQ with an increase in culture duration as well as deficiency of either of the nutrients. However, the addition of extra bicarbonate rendered stress amelioration effects.

Semi-quantitative Gene Expression Profiling of Selected Enzymes

Figures 6A-C represented the differential expression of accD, rbcL and rbcS genes at 0, 6, and 12th d in the selected cultures of D. salina. ACCase, the enzyme responsible for catalyzing the first and rate-limiting step of lipid biosynthesis, has been the point of interest for the genetic engineering perspective [59]. Thus, optimization of lipid over-production strategies must be accompanied by intricate evaluation of accD (the chloroplastidic subunit of ACCase) expression in such conditions. Nitrate-deprived cultures of D. salina triggered 1.84-fold surge in the expression of accD at the 6th d (p < 0.05), but only 1.62-fold at the 12th d. Post hoc analysis revealed that the change in accD level in –N cultures was significant between 0 and 6th d (p < 0.05) and non-significant between 6 and 12th d, indicating the triggering of accD gene expression in the earlier stage under –N condition. Conversely, under Nlim condition, maximum expression (2.23-fold) was monitored at 12th d against 1.14-fold at the 6th d (p < 0.05 between 6 and 12th d) indicating triggering of lipid synthesis at a relatively later stage in Nlim cultures with greater vigour. Nlim cultures afforded the acceleration of lipid production beyond the 6th d (since nitrogen remained available for a longer period). These data were in tune with the temporal variations in the NRF levels of the respective cultures. The data are supported by the findings of [60] who had shown significant up-regulation of accD expression in nitrate-limited C. sorokiniana cells. Similarly, Singh and co-workers found 4.4-fold increase in accD expression in C. sorokiniana cells cultured under limited nitrate and phosphate simultaneously [16]. It was also clear from the data that nitrogen deficient media and even its augmentation with that of C2 (10 mM HCO3 −) didn’t favour the expression of rbcL and rbcS genes as was evident from post hoc analysis (p = 0.243 and 0.993 for rbcL and rbcS at the 12th d respectively compared to control). This reduction in BMPr may be explained in the light of reports of [61] that inferred that nitrogen deprivation conditions inflicted reduced activities of RuBisCO, tryptophan synthase (β-subunit) and 16S ribosomal subunit. This led to inefficient photosynthesis, low amino-acid turnover, and a subsequent reduction in BMPr. Nutrient starvation in another green alga C. pyrenoidosa also negatively affected RuBisCO function as rbcL expression was down-regulated by 3–5 fold [62].

Fig. 6
figure 6

Differential expressions of (A) accD (first and rate limiting gene of lipid biosynthesis), (B) rbcL and (C) rbcS (involved in inorganic carbon fixation) genes in relation to varying culture regimes at three different time points. Vertical bars represent mean value ± SD of triplicate readings. Different letters suggest means are significantly different (Tukey’s test; p < 0.05). Upper case letters correspond to significant differences in folds of gene expression at different time points within the same nutrient treatment while lower case letters signify significant differences in relative gene expression between different nutrient conditions at the same time points

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Interestingly, C2 addition to Nlim culture significantly raised the larger and smaller subunits of the RuBisCO on the 12th d by 2.53 and 1.78-fold respectively (p < 0.05) indicating that a critical threshold limit of nitrogen was necessary for over-expression of RuBisCO subunits for the assimilation of abundant inorganic carbon. Such cultures with a critical threshold limit of nitrogen and deficient in phosphate (Nlim–P) were recorded with 3.23-fold increase in accD gene transcripts compared to the control (p < 0.05). The addition of C2 resulted in the highest level of accD expression (6.23-fold, p < 0.05) which might be due to the concerted action of the cells to channelize all the resources towards lipid production as the phosphate deficiency triggered it from the beginning. A study on another algal species, C. sorokiniana has shown 9.6 and 2.8-fold enhancement in expression of accD and rbcL under combined stress involving metal and nutrients [16]. Expression of rbcL in Nlim–P remained higher and equivalent to the Nlim cultures, indicating that the phosphate deprivation had little impact on carbon capture in the D. salina cells. Therefore, it can be inferred that carbon augmentation to Nlim-P cultures may be adjusted accordingly for accD over-expression. As far as our knowledge goes, this is the first report in D. salina deciphering the critical threshold limit of carbon and nitrogen level in culture media coupled with the expression profiling of key genes involved in biomass and lipid accumulation to achieve higher levels simultaneously.

Conclusion

Overproduction of the lipid in nutrient(s) deficient growth media suffers from lower biomass productivity in D. salina. Among all the tested combinations of selected nutrients, limited nitrogen supplementation (1.25 mM), phosphate starvation along with augmentation of bicarbonate (10 mM), led lipid accumulation (~ 40% DCW) in D. salina with significant increase in biomass (27%) also, compared to the control. Such cultures were recorded with up-regulation of rbcL and rbcS as well as accD genes, indicating enhanced carbon flux in the cells. These cells showed photosynthetic quantum yield (> 0.5) reflecting healthy status. Results outlined the basis of selecting applicable nutrient stress condition for mass cultivation of D. salina. The over-expressed genes may act as target(s) for hyper production of lipids using genetic engineering approaches. Thus, the halophilic D. salina, a treasure of high value compounds including lipids must be studied in a well-defined manner for application prospective in industrial sector.