The growth of microalgae depends on an adequate supply of nutrients mainly nitrogen, phosphorus and micronutrients. Nitrogen (N) is a major component in many biological macromolecules like chlorophylls, proteins and DNA. Under N depletion, microalgae grow in a medium lacking of N source, while under N limitation there is a constant but insufficient N availability. Therefore, the N nutrient stress on cellular physiology negatively affects microalgal growth such as cell density and dry biomass (Benavente-Valdés et al. 2016; Ördög et al. 2012).
The P. salinarum growth response was proportional to the N concentration in the medium during the culture period (20 days). The control culture (1 N) reached the highest values of cell density from the 18th day until the end of culture period on the 20th day (13.1 x 106 cells mL-1), and a gradual decrease in the population growth with the increase of nitrogen limitations (0.5, 0.25 and 0.125 N) (Fig. 1). These ﬁndings corroborate the results obtained by other researchers, who reported that microalgae cell density is directly proportional to the concentration of N in the culture medium (Chen et al 2011; Dean et al 2010; Illman et al 2000; Zhu et al. 2014).
Regarding the biomass yield through the dry weight (Fig. 2), the highest values were obtained in 1 N (0.96 g L-1) and 0.5 N (0.88 g L-1) on the 20th day. They were followed by the dry weight decrease in response to N limited availability under 0.25 and 0.125 N treatments. Similar studies in other green microalgae showed the dry mass decreased under N limitation, from 2.88 g L-1 to 0.97 g L-1 in Chlorella minutissima (Ördög et al. 2012) and from 1.17 g L-1 to 1.05 g L-1 in Dunaliella salina (Sathasivam et al. 2018). Under N depletion conditions, high dry weight decrease values from 1.39 g L-1 to 0.06 g L-1 was reported for Scenedesmus quadricauda (Anand and Arumugam 2015).
N limitation decreased the chlorophyll content of P. salinarum (Fig. 3 a, b). Chlorophyll a content showed the highest values in the control since the 12th day and reached 2.73 µg mL-1 on the 20th day. However, a sharp dropped of 91.2% in chlorophyll a content was evidenced under the 0.125 N treatment by the end of the culture period. High chlorophyll b content was obtained from the 16th day to the 20th day (0.92 µg mL-1) in control culture. A high decrease of chlorophyll b (84%) was notorious under nitrogen limitation (0.125 N treatment). On the opposite, P. salinarum N limitation was related positively with the carotenoids content. The highest production started on the 12th day with a gradual increase up to 3.35 µg mL-1 on the 20th day in 0.125 N treatment, that is seven times higher than algal growth in the control culture (Fig. 3 c).
The photosynthetic pigments of the 0.5 and 0.25 N treatments showed intermediate values between 1 and 0.125 N treatments throughout the culture period. Then, the chlorophyll content was related positively to the nitrogen levels tested. However, the carotenoid accumulation was related negatively to the nitrogen levels. These results recognized in the culture flask coloration with greenish pigmentation at the beginning, later with time the cultures with normal nitrogen supply had an intense green color, however, those with the lowest nitrogen supply changed to a yellowish coloration.
Similar results were recorded in Dunaliella salina, with a chlorophyll content decreased from 27.90 μg mL-1 to 10.20 μg mL-1, when the nitrogen concentration was reduced in half. Conversely, the carotenoid content increased from 99.43 μg mL-1 to 177.10 μg mL-1 (Sathasivam et al. 2018). Chlorophylls decreased and carotenoids increased in culture under N stress conditions were reported for the green freshwater microalgae Chlamydomonas reinhardtii (Cakmak et al. 2012) and Dunaliella tertiolecta (Young and Beardall 2003).
Therefore, there was a progressive loss of certain plastid functions, with impact in photosynthetic pigments such as the decrease in chlorophyll synthesis and an increase in carotenoids with the limiting nitrogen nutrient. This occurrence is related to the reorganization of the photosynthetic apparatus to maximize the efficiency of absorption of specific spectra of light under situations of nutritional stress (Young and Beardall 2003). Chlorophyll is a nitrogen-rich compound utilized as an intracellular nitrogen pool to support cell growth. Then, chlorophyll concentration would account for the cell density increase and biomass production registered in the control culture (75 mg L-1) and 0.5 N treatment (37.5 mg L-1).
Differential responses in total protein, carbohydrate, and lipid contents of P. salinarum under N limitation culture treatments are showed in Table 1. Nitrogen is an essential element for amino acid synthesis, its deficiency reduce dramatically protein biosynthesis, trigger the inhibition of citric acid cycle and a drastic cell division decrease due to protein reduction in the photosystem reaction center and photosynthetic electron transport (Deng et al. 2011; Msanne et al. 2012).
The total protein content of P. salinarum under 0.25 and 0.125 N treatments showed a remarkable decrease of 78.08 mg g-1 related to 7.81% DW and 18.92 mg g-1 related to 1.8 % DW, respectively (Table 1). Cobos et al. (2017) reported a decrease in protein content under N depletion for the freshwater microalgae species as follows: Acutodesmus obliquus from 12.8% to 9.7% DW, Ankistrodesmus sp. from 14.5% to 10.5% DW and Chlorella lewinii from 31.2% to 14.2% DW. Dean et al. (2010) reported an 18% decrease in the protein content of Chlamydomonas reinhardtii in response to N limitation. However, Cakmak et al. (2012) cited a notorious reduction up to 82% in this species.
Under N depletion or N limitation, alternative metabolic pathways for fixing inorganic carbon such as the synthesis of carbohydrates or lipids in microalgae are activated (Deng et al. 2011; Msanne et al. 2012; Pancha et al. 2014). The carbohydrates production is mainly related to the cell wall structural components and nutritional reserves (Markou et al. 2012). Our work demonstrated that carbohydrate content was the main biochemical fraction for cultures with high nitrogen concentrations: 1 N and 0.5 N with 43.14% and 43.56% DW, respectively. However, the low carbohydrate contents were obtained in P. salinarum grew under 0.25 and 0.125 N treatments with 34.48% and 30.98% DW, respectively. Therefore, under N extreme stress conditions that is 9.375 mg L-1 NaNO3 (0.125 N), carbohydrate content decreased and became the main second biomolecule followed by the lipid content (Table 1).
The effects of N limitation on the carbohydrates accumulation in cultures reported for Chlorella vulgaris represented 41% DW (Dragone et al. 2011), 35% DW in Tetraselmis subcordiformis (Yao et al. 2012) and 57% DW in Desmodesmus sp. (Sanchez Rizza et al. 2017). However, Chlamydomonas reinhardtii increase up to its 80% DW (Cakmak et al. 2012; Siaut et al. 2011).
The lipids have a main role in cell membrane structural composition. Nevertheless, under nutritional limitation, due to their hydrophobic nature, lipids are derived as a storage product. They present very low states and are efficiently packaged in the cell and can be used under adverse conditions for survival and subsequent cell proliferation (Courchesne et al. 2009). The increase in total lipid content could be explained for a boost in transcript levels of genes encoding enzymes of the lipid biosynthesis pathways, specifically in the last step in the Kennedy pathway of triacylglycerol biosynthesis (Deng et al. 2011; Weiss et al. 1960).
In the present study, the N limitation caused an increase in the lipid content of P. salinarum, reaching the highest value of 33.87% DW under 0.125 N treatment, becoming the main biomolecule. The lipid content increases in variable ways in other algae species, then in Nannochloropsisoceanica almost duplicate with increase from 7.9% to 15.31% DW, and in Chlorellavulgaris from 5.9% to 16.41% DW (Converti et al. 2009). Other species of Chlorella, C. emersonii and C. minutissima showed a high increase of lipids in the order of 63% and 56% DW, respectively (Illman et al. 2000). However, in C. lewinii there was an increase from 9.5% to 13.2% DW, in Acutodesmus obliquus from 15.2% to 18.8% DW and in Ankistrodesmus sp. from 23.7% to 39.5% DW (Cobos et al. 2017).
The results previously described suggest that depending on the N concentration supplied and the type of species, the microalgae synthesize a certain biomolecule to face the nutritional deficit and continue with its development. In this context, the organic carbon generated by photosynthesis is related to the biomolecules production such as carbohydrates and lipids. They are storage in reserve subcellular structures and accumulated at expense of reduced growth rate (Msanne et al. 2012; Siaut et al. 2011).
In addition, the TEM analysis of P. salinarum vegetative cells under different N concentrations showed cell structural changes (Figure 4). Under normal conditions, longitudinal sections of cells showed oval shape with a typical chloroplast occupying most of the cell volume (Fig. 4 a). This observation was similar to TEM images recorded for same microalgae species (Glabonjat et al. 2020; Lopes Dos Santos et al. 2017). P. salinarum under nitrogen depletion treatments accumulated organic material reserve as starch grain and lipid droplet (LD) (oil body or oleosome) in several numbers and sizes for each treatment (Fig. 4 b, c, d).
It has been proposed that the lipids are synthesized and packaged initially in the plastid and then transported to the cytoplasm, where they form the LDs (Eltgroth et al. 2005). These structures are the main storage structure for neutral lipids in eukaryotic cells, and support evidence they are involve in other cellular processes such as lipid homeostasis and communication signaling between other organelles. The LD synthesis in response to specific cellular needs and their number per cell change according to the nutritional status conditions (Goold et al. 2015). Also under N limitation or N depletion conditions, both the number and size of the LDs can increase and the chloroplast became imperceptible, because they act as a sink for membrane-derived fatty acids, including plastid membrane lipids that are degraded (Goold et al. 2015; Roopnarain et al. 2014; Siaut et al. 2011).
The biochemical composition of P. salinarum was in agreement with our TEM results. In 0.5 N treatment, several starch grains dispersed were observed compared to LDs, and the chloroplast was hardly visible (Fig. 4 b). However, under 0.25 N treatment, the large LDs development was notorious as well as starch grains decreased (Fig. 4 c). Furthermore, at 0.125 N treatment, a dominant single LD occupied most of the cell volume as well as several small ones around it (Fig. 4 d).
Under normal growth conditions, Chlamydomonas reinhardtii has a single cup-shaped plastid that occupied more than two-thirds of the total cell volume, in some strains neither starch grains nor lipid droplets were detected. The appearance and accumulation of these reserve structures, as well as the reduction of plastid organelle, were notorious in cells under N depletion (Siaut et al. 2011). Zhu et al. (2014) observed in Chlorella zofingiensis an increase in starch grains both in size and number after the first days under N stress, with a few LDs. Through the coming days, the cells exhibited more LDs instead of starch granules. Then, small LDs fusion formed larger ones. Other studies reported that the starch granules can fuse and be converted into LDs, this analysis suggests that the carbon flux of starch must provide some of the precursors for lipid synthesis (Ito et al. 2013; Mizuno et al. 2013).
These findings were in agreement with our results and suggested the presence of a single large LD in P. salinarum cells under the high N limitation tested (0.125 N treatment), as storage lipid product probably became greater as carbon source (starch grains) were useful during the algal growth period.
Profile of fatty acid methyl esters (FAMEs)
The determination of the FAMEs profile as well as the biomolecule concentrations related to their proportion with the dry weight are essential steps for characterizing microalgae strains as potential raw material source for biodiesel. The FAMEs analysis with the profile of saturated fatty acids (SFAs) and unsaturated fatty acids (UFAs) of P. salinarum under N limitation is presented in Table 2.
Among SFAs, the lauric acid (C12:0) content had a proportional reduced response to the supply decrease of N and not being detected at 0.125 N treatment. In contrast, palmitic acid (C16:0) and myristic acid (C14:0) increased their contents. Under the highest N limitation (0.125 N), the C16:0 content reached a high value of 923.95 µg g-1 DW. Regarding to the UFAs, oleic acid (C18:1) also had a decrease content in response to the lowest N supply in cultures. In addition, linoleic acid (C18:2) and linolenic acid (C18:3) reported minimum values or were not detected in all treatments.
It is known that N limitation stimulates the C16:0 production between the SFAs, as it were reported for green microalgae such as Chlorella vulgaris, Nannochloropsis oculata, Chlamydomonas reinhardtii, Dunaliella salina and Dunaliella tertiolecta (Chen et al. 2011; Converti et al. 2009; Lv 2016; Msanne et al. 2012). In our studies, the decrease in UFAs mainly C18:2 and C18:3 due to N limitation was correlated with those registered for Coccomyxa sp. (Msanne et al. 2012). Besides, Anand and Arumugam (2015) reported that the main fatty acid detected (C18:1) in Scenedesmus quadricauda had a drastic reduction under N limitation. On the other hand, this fatty acid was not synthesized in some algae such as Dunaliella salina (Lv 2016). Therefore, it is confirmed that the microalgae cells tend to decrease the degree of fatty acid unsaturation in response to the N deficit.
The FAMEs profile plays an important role in the biodiesel quality. It determines its viscosity, lubricity, total unsaturation (iodine value), density, oxidative stability, cetane index (ignition quality indicator), cold flow property and calorific value (Francisco et al. 2010; Knothe 2005). The FAMEs of the microalgae are different from those of higher plants, the last are special rich in polyunsaturated fatty acids (PUFAs) such as C18:2 and C18:3. These have four or more double bonds, being more susceptible to oxidation during storage which reduce its acceptability for use in biodiesel use. Besides, as the PUFAs concentration is high, the biodiesel nitrate and nitrite emission rate increases (Chen et al. 2018; Chisti 2007; Francisco et al. 2010).
On the other hand, when the SFAs are high, they result in a lower cetane index and increase the biodiesel stability since the SFAs are more resistant to auto-oxidation (Knothe 2005). Guidelines international and regional control of oily biomass for biofuel use; like the requirements of the European Norms EN 14213 and EN 14214, pointed out that the C18:3 amount must have a limit lower than 12% of the total FAMEs for motor vehicles use (Knothe 2006). By this way, biomass highly rich in oils with high levels of saturated fatty acids is sought, and meets local criteria for use and biofuel production.
The PCA provided an overview of the N limitation effects on P. salinarum (Fig. 5). Its biochemical composition under control conditions (1 N) had the expected results in green microalgae, high growth (cell density and dry mass) was related to high contents of chlorophyll a and b, proteins and carbohydrates. The same patterns were also followed with the 0.5 N treatment. Besides, the degree of fatty acids saturation was recognized under the 0.25 and 0.125 N treatments, mainly with the last treatment related to the lipid content, carotenoids and C16:0. P. salinarum biomass (0.125 N treatment) with high total lipid yield and adequate fatty acid composition that is high SFA content (C:16), and a low C18:3 (PUFA) met certain European Norms requirements. Therefore, it showed to be a competent and potential raw material source for the biodiesel production.