Effect of Nitrogen Limitation on Growth, Biochemical Composition and Cell Ultrastructure of the Microalga Picocystis Salinarum

In recent years, biodiesel production has attracted worldwide attention due to the awareness of fossil fuel depletion, and microalgae biomass is considered a promising raw material for its formulation. The present study evaluated the effects of different levels of nitrogen limitation (37.5, 18.75, 9.375 mg L -1 NaNO 3 ) on the growth, cell ultrastructure and biochemical composition of Picocystis salinarum as a potential raw material source for biodiesel. During a culture period of 20 days, the growth measurements were estimated, and cell density, dry weight and chlorophylls a, b content decreased with time as nitrogen limitation increase, however, carotenoids content increased. The high N limitation (9.375 mg L -1 ) had a highly signicant effect on the accumulation of total lipid content (33.87% dry weight), carbohydrate content increase (30.98% dry weight), but protein content decrease (1.89% dry weight). The lipid content showed a differential FAME prole with high saturated fatty acid values (996.08 µg g -1 dry weight) mainly C16:0, compare with the unsaturated ones that showed low values under high N limitation. The gradual increase of lipid content was also corroborated by transmission electron microscopy images with lipid droplet cell formation. Therefore, evaluation of the algal culture conditions such as N limitation, as a strategy to maximize lipid content and improve the fatty acid prole in unexplored halophilic P. salinarum showed a potential biomass yield as a suitable candidate for biodiesel production.


Introduction
The common use of petroleum-based fuels is widely recognized as unsustainable, it has been considered a global concern due to the exhaustion of its stocks, and the huge emission of greenhouse gases into the atmosphere contribute to climate change (Tan et al. 2018). Between, substitute alternatives to petroleum products (diesel fossils) the biodiesel has become a potential renewable fuel and its use leads to a reduction of harmful carbon dioxide emissions and the elimination of sulfur oxide emissions (Francisco et al 2010).
Biodiesel could be derived from food crops as edible oilseeds (sun ower, palm, soy, coconut), considered rst-generation raw materials. However, mass production of alternative fuel source as terrestrial oil crops may cause shortage for food supply as well as deforestation (Chen et al. 2018; Rawat et al. 2013;Tandon and Jin 2017). Recently, non-food crops (non-edible seeds: pine nut, karanja, jojoba, mahua), edible oils residues, and animal fats, have gained importance as second-generation raw materials. Nevertheless, they do not have enough lipid content to replace biofuel current needs (Rawat et al. 2013; Tandon and Jin 2017).
In this context, microalgae emerged as biodiesel source to cover global demand for fuel due to their high growth rate, photosynthetic e ciency, and high lipid content biosynthesis (Chen et al. 2018;Chisti 2007). Consequently, microalgae biomass yield is considered as the third generation raw material for biodiesel.
Their use does not interfere with food production and competition for arable land is reduced, and the water volume requirement is much lower for their biomass production compare to cultivable plants in the agronomic activity. Besides, certain microalgae biomass contains other biomolecules including carbohydrates, proteins and pigments that can be used for different secondary value-added products such as food, pharmaceutical or cosmetic additives (Demirbas and Demirbas 2011;Tandon and Jin 2017).
Microalgae can be grown in several synthetic media based on freshwater or seawater. To avoid competition for freshwater and signi cantly contribute to the biodiesel economy from microalgal biomass, the selection of a cultivable strain in seawater is mandatory (San Pedro et al. 2013). Although many oleaginous microalgae have already been studied, there are a large number of unexplored species, mainly from extremophile continental aquatic ecosystems (acidophilic, alkaliphilic, halophilic or thermophilic), which may have an even greater potential as a source of grease raw material for biodiesel production (Malavasi et al. 2020;Sanchez Rizza et al. 2017). Furthermore, it is necessary to manipulate the biochemical composition of the strain to increase their lipid content with synthesis of speci c fatty acids for biodiesel formulation by adjusting the nutrient composition, salinity or pH of the media, and varying culture conditions such as light, temperature or photoperiod (Juneja et al. 2013).
Nutrient depletion is an approach to target metabolic pathways in lipid synthesis as the main reserve substance in microalgae. Although it has been reported that phosphorus and iron channel the metabolic ux to lipid biosynthesis under normal conditions, nitrogen is considered the most effective nutritional limiting factor for triggering high oil contents (Courchesne et al. 2009). Between the green microalgae, the halophilic Picocystis salinarum had registered intracellular lipid droplets during its cultivation under nitrogen limitation ).
Therefore, the present research aimed to evaluate the effect of nitrogen limitation as a strategy to increase the lipid and fatty acid productivity of the biomass of P. salinarum as a potential raw material for biodiesel. In addition, analysis of changes in biochemical composition, growth and cell ultrastructure of this microalga is reported.

Strain and culture conditions
The Picocystis salinarum strain USM 303650 was obtained from the Herbarium of the Natural History Museum, National University of San Marcos, and its culture was carried out at the Laboratory of Taxonomy and Ecology of Algae Continents, Federal University of Espírito Santo.
Batch cultures were grown in 3 L Erlenmeyer asks (2.7 L culture medium and 0.3 L of axenic inoculum with 9.5 ± 0.5 x 10 5 cells mL -1 ) for twenty-days. Lyophilization was set up to obtain microalgal biomass.

Experimental design
Cultivation experiments were conducted to evaluate and compare the algal growth, cell ultrastructure and biochemical composition of the P. salinarum strain under different nitrogen limitation conditions. The original sodium nitrate concentration (75 mg L -1 ) in f/2 medium is reported as 1 N (normal conditions).

Growth assessment
Cell density (10 6 cell mL -1 ) was determined by direct counting in a Neubauer hemocytometer under an optical microscope (Olympus, CX41, Japan), every two days during the culture period (20 days). Dry weight (g L -1 ) was calculated by gravimetry every four days, culture aliquots (20 mL) were ltered in a glass ber micro lter (Macherey Nagel, GF-1 47 mm, Germany), followed by over drying at 60 °C until constant weight.
Quanti cation of photosynthetic pigments Chlorophylls a, and b, as well as total carotenoids were extracted in 90% acetone and measured every four days in a spectrophotometer (Thermo Scienti c, AquaMate Plus, USA). Pigments concentration (µg L -1 ) were calculated according to the equations proposed by Jeffrey and Humphrey (1975) and Strickland and Parsons (1968), respectively.
Determination of protein, carbohydrate and total lipid Total protein content was determined according to Lowry et al. (1951). For alkaline hydrolysis, 4 mL of 1 N NaOH was added to 5 mg of microalgal biomass, and the mixture was incubated at 100 °C for 1 h and centrifuged at 3000 rpm for 30 min. Then, 5 mL of solution of 2% Na 2 CO 3 in 0.1 N NaOH, 0.5% CuSO4 and 1% KNaC 4 H 4 O 6 (100: 1: 1, v/v/v) was added to 0.5 mL aliquot of the alkaline extract. The mixture was kept for 10 min at room temperature. Then, 0.5 mL of Folin-Ciocalteau reagent in distilled water (1: 1, v/v) was added and the mixture was incubated for 30 min. The blue complex was analyzed in a spectrophotometer set at 750 nm against a calibration curve of albumin solution of known concentration as the standard.
Total carbohydrate content of the microalgal biomass was determined using the phenol-sulfuric acid method proposed by Kochert (1978). For this purpose, 5 mg of biomass was tested to alkaline hydrolysis. Afterward, 1 mL of 1 N NaOH and 0.05 mL of 4% phenol were added to 0.5 mL aliquot of alkaline extract. The mixture was kept in an oven at 25 °C for 30 min. Then, 2.5 mL of sulfuric acid was added to the mixture and was kept for 5 min at room temperature. The yellow-brown complex was analyzed by spectrophotometric analysis at 485 nm against a calibration curve (anhydrous glucose solution).
Total lipid was extracted according to Bligh and Dyer (1959). 7.5 mL chloroform and methanol solution (1: 2, v/v) was added to 0.5 g biomass. The mixture was vortexed for 2 min followed by addition of 2.5 mL chloroform and 2.5 mL distilled water, and vortexed again. Subsequently, the mixture was centrifuged at 3500 rpm at 4 °C for 8 min. The organic phase with the extracted lipids was separated and placed in an oven at 30 °C for organic solvent evaporation. Finally, the total lipids were calculated gravimetrically.
The total protein, carbohydrate and lipid concentrations are given as mg g -1 and biomass dry weight (% DW).

Transmission electron microscopy (TEM)
Cell ultrastructure was studied using the TEM which was performed according to the method of

Analysis of fatty acid methyl esters (FAMEs)
Fatty acids were determined using high-performance liquid chromatography (HPLC) of methyl esters from microalgal biomass. First, the transesteri cation of biomass was performed according to Menezes et al. (2013) with few modi cations. 2 g of biomass was suspended in 3 mL of 0.5 M NaOH in methanol, followed by heating at 70 °C for 10 min. Then, samples were cooled in an ice-water bath and added 9 mL of an esterifying solution of NH 4 Cl: methanol: H 2 SO 4 (1: 30: 1.5, g/v/v). Samples were again heated at 70°C , cooled in an ice-water bath, and 5 mL heptane and 2 mL distilled water were added to the mixture and vortexed. The heptane phase containing FAMEs were transferred into a tube and dried under a stream of nitrogen.
A binary mobile phase consisting of (A) tri uoroacetic acid solution and distilled water (0.1: 99.9, v/v) and (B) acetonitrile were ltered using a vacuum ltration system through 0.45 μm membrane lters and degassed in an ultrasound bath (Limpsonic, Brazil). The HPLC system was programmed to operate under controlled conditions of column temperature (37 °C), detection wavelength (210 nm) and ow rate (0.25 mL min -1 ). The following gradient elution was employed: 0 -1 min: 100% A; 1 -12 min: 90 -70% A, 20 -40% B; 12 -32 min: 100 -90% A, 62 -40% B; 32 -32.5 min (column equilibration): 100% A. The fatty acids were analyzed by comparing their retention time of the corresponding peaks with a known standard mixture of FAMEs added to each sample as the standard. LCSolutions 2.1 software was used for data acquisition and analysis.

Statistical analysis
The tests were performed using triplicates for each treatment. Means and standard deviation (SD) were calculated for all treatments, and signi cant differences were determined by analysis of variance according to Tukey's highly signi cant differences test (p < 0.05). Comparison among the treatments was performed by one-way ANOVA test (p < 0.05). Principal component analysis (PCA) was used to determine the relationship between all tests analyzed. ANOVA and Tukey's test were performed using the SPSS 20.0 software, and the PCA using the XLSTAT 2020 software.

Results And Discussion
Growth measurements 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 insu cient N availability. Therefore, the N nutrient stress on cellular physiology negatively affects microalgal growth such as cell density and dry biomass (Benavente-Valdés 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 20 th 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).
Photosynthetic pigments 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 12 th day and reached 2.73 µg mL -1 on the 20 th 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 16 th day to the 20 th 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 12 th day with a gradual increase up to 3.35 µg mL -1 on the 20 th 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 ask 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. 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 e ciency of absorption of speci c 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 ).

Biochemical composition
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 de ciency 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) 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).

Cell ultrastructure
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 de cit 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 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 zo ngiensis an increase in starch grains both in size and number after the rst 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 ux of starch must provide some of the precursors for lipid synthesis (Ito et al. 2013;Mizuno et al. 2013).
These ndings 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.
Pro le of fatty acid methyl esters (FAMEs) The determination of the FAMEs pro le 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 pro le 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 con rmed that the microalgae cells tend to decrease the degree of fatty acid unsaturation in response to the N de cit.
The FAMEs pro le 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 ow property and calori c 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.

Conclusions
It is known, that the raw material composition for biodiesel, must be rich in lipids with certain group of fatty acids. The majority of higher plants and microalgae biomass have considerable contents of proteins and carbohydrates, but inadequate fatty acids composition, therefore, they would not be useful as a raw material for biofuel industry. To solve this performance problem, the approach developed in this work (decrease in the N supply in culture) reported the e ciency of lipid content with an adequate fatty acid pro le in the halophilic extremophile P. salinarum. A reduction in the protein and carbohydrate content, but an increase of lipids under the high N limitation (0.125 N) was showed for P. salinarum biomass. The fatty acid pro le obtained is an advantage due to the proportion between SFAs and UFAs for a suitable biodiesel. This also was supported with the TEM micrographs of cell cultures under stress N limitation with a single large-volume LD that suggested a large scale oil extraction would be performed successfully.
The ndings of this research also suggest that P. salinarum biomass is a potential source of grease raw material suitable for the production of biodiesel, which could contribute to sustainable development as a viable alternative to petroleum exploration. However, it will be necessary to develop cultivation systems for biomass production on a large scale for biofuel production and additional bioactive compounds    Figure 1 Effect of nitrogen limitation on the cell density of P. salinarum. Data are expressed as mean ± SD (n = 3).

Figures
Values with the different letters represent signi cant difference (p < 0.05) between treatments.

Figure 2
Effect of nitrogen limitation on the dry weight of P. salinarum. Data are expressed as mean ± SD (n = 3).
Values with the different letters represent signi cant difference (p < 0.05) between treatments.