## 3 Results and Discussion
### Synthesis of Storage Lipids and Fatty Acid Composition of Total Lipids and Lipid Fractions  
The lipid content was at similar levels for _P. octavermella_ VAS2.5 (L/x% = 18.5 \(\pm\) 1.4%, wt/wt) and _M. gaditana_ VON5.3 (L/x% = 18.3 \(\pm\) 2.3%, wt/wt). _P. oklahomense_ SAG4.4 also exhibited satisfactory lipid accumulation, though lipid reserves were decreased from L/x% = 23.2 \(\pm\) 0.9% (wt/wt) at 10 days to L/x% = 13.2 \(\pm\) 1.1% (wt/wt) by the end of the culture. This phenomenon has been observed elsewhere and was attributed to the reduction in photosynthesis due to the increase in cell density of the culture [24]. Thus, cells turned to the reserves, which can be used for energy conservation and/or further growth. The lowest lipid accumulation rate was recorded for _N. pyriformis_ PAT2.7, which was L/x% = 7.3 \(\pm\) 0.3% (wt/wt). In general, these results are very encouraging and indicate the potential of these newly isolated strains to produce even higher yields. Furthermore, it must be taken into account that the microalgae were grown on mASW, which was neither nitrogen nor phosphorus-limited (Table 2). Nitrogen and/or phosphorus limitation or absence from the growth medium usually triggers lipid accumulation [24]. Other influential factors for lipid content are temperature and light intensity, though in a species  
Figure 3: Morphology of cells of the four microalgal strains under an optical microscope after their cultivation in mASW. (**a**) _Picochlororum costavermella_ VAS2.5; (**b**) _Picochlororum oklahomense_ SAG4.4; (**c**) _Microchloropsis galitana_ VON5.3; and (**d**) _Nephroselmis pyriformis_ PAT2.7. Total magnification: 400\(\times\). and strain-dependent manner [45]. For example, when _N. oculata_ was cultured in an ORWP containing modified F/2 medium with ASW that had as a nitrogen source NaNO\({}_{3}\) 0.25 g/L (i.e., 0.18 g/L of nitrate) instead of KNO\({}_{3}\) 1 g/L (i.e., 0.61 g/L of nitrate) of this study (Table 1), lipid accumulation ranged from 26 \(\pm\) 1% to 44 \(\pm\) 4% (wt/wt) and from 42 \(\pm\) 5% to 44 \(\pm\) 7% (wt/wt) during the winter and spring seasons, respectively [6]. Another important parameter affecting lipid biosynthesis is the growth phase of culture. For instance, the biomass of _N. oceanica_ was mainly composed of protein and carbohydrates, with relatively low lipid contents at the late exponential phase [5]. According to the authors, low lipid contents suggested that the cells' growth occurred in the absence of nutrient stress, which was correlated with the physiological phase of the culture. Herein, as shown in Figure 2, most of the strains used at the time of biomass harvesting were in the exponential or late exponential phase. Additionally, in Table 2, the percentages (%, wt/wt) of lipid fractions relative to total lipids are presented. The predominant lipid fraction was that of G+S (43.6-55.4%, wt/wt), followed by NL (27.1-50.1%, wt/wt) and finally P (6.9-17.4%, wt/wt). The predominance of the polar structural lipids (i.e., G+S and P) over the main storage neutral lipids was anticipated since it seems to be a common feature for many microalgae of various genera, like _Chlorella_, _Tetraselmis_ (Chlorophyta), and _Nannochloropsis_ (Eustigmatophyceaeae) [24; 46]. The above-mentioned can be answered by the fact that glycolipids and phospholipids are the major components of chloroplast and thylakoid membranes, while sphingolipids constitute a diverse group of membrane lipids of high importance for various cell functions, including the control of cell division or communication with the cell's environment [47; 48]. Both G+S and P lipid fractions are considered important sources of omega-3 fatty acids, mainly of \(\Delta\)9,12,15C18:3, \(\Delta\)58,11,14,17C20:5, and \(\Delta\)47,70,13,16,19,C22:6. PUFAs, due to their exceptional structure, possess a key role in the regulation of membrane fluidity and function [49; 50; 51; 52].