## 3 Results and Discussion
### Fatty Acid Methyl Esters Analysis (FAMEs)  
Polyunsaturated fatty acids (PUFA) were present in greatest abundance, followed by monounsaturated fatty acids (MUFA) and lower amounts of saturated fatty acids (SFA), which constituted 41.86%, 31.80%, and 26.34%, respectively (Table 3). MUFA levels were lower in RS (28.91%) compared to W (31.80%) or R (31.85%) (Table 3). In addition, the _P. tricornutum_ PUFA ratio of omega-3/omega-6 was lower in R and RS compared to control W (Table 3). The evidence suggests that a high ratio of dietary omega-3/omega-6 PUFAs reduces the risk of several diseases [52]. Therefore, _P. tricornutum_ cultured in W is a potentially more valuable source of functional food and animal feed components than _P. tricornutum_ cultivated under R or RS conditions. These results suggest that the RS condition regulated the fatty acid content in _P. tricornutum_, specifically lowering MUFAs and increasing PUFAs. It has been reported that stress conditions such as nitrogen depletion, high and low temperature, and continuous high light intensity impacts fatty acid composition, specifically PUFA in _P. tricornutum_[53; 54; 55]. The distribution of fatty acids was characterized (Appendix A; Table A1), and various lipid classes of _P. tricornutum_ are summarized in Table 4. The main lipid components were mid-chain FAs, 14:0, 16:0, 16:1n-7, and 20:5n-3, which together represented 76.46% of total fatty acids (Table 4). Our results are similar to those reported by [42]. Palmitoleic acid (16:In-7), which constituted 28.20% of the total FAs, was predominant in _P. tricornutum_ control (W) but was reduced to 24.29% in RS condition (Appendix A; Table A1). Interestingly, hexadecatrienoic acid (16:3n-4) was increased in R and RS compared to W (Appendix A; Table A1). There was a comparable amount of the omega-3 fatty acid, eicosapentaenoic acid (20:5 n-3), in all light conditions and it constituted almost 22% of total acids, which was the second most predominant FA (Table 4). In other studies, it was the PUFA that was present in highest mass proportions [53; 54; 55]. Humans do not produce sufficient long-chain omega-3 fatty acids to meet their physiological needs; therefore, it is essential to obtain them from external sources. Omega-3 fatty acids have been associated with proper fetal development, including neuronal, retinal, and immune function [56]. They have also shown positive effects on coronary disease and inflammation, and they have been approved for the treatment of patients with severe hypertriglyceridemia [33]. These fatty acids are predominantly found in cold water fish, such as salmon. However, our work suggests that microalgae and diatoms can be considered as a sustainable and vegetarian source of omega-3 fatty acids. The structural features (chain length, unsaturation, and branching) of fatty acids determine the properties of algae-derived biodiesel, including ignition quality, heat of combustion, cold flow, oxidative stability, exhaust emissions, viscosity, and lubricity [57]. Thus, the major fractions of fatty acid composition, which was accepted as input in the Biodiesel Analyzer, were studied for properties of biodiesel [23] (Table 5). For biofuel of an industrial quality, it needs to have similar physical properties as diesel, which can be used efficiently in combustion engines. According to a study, fatty acids with chain lengths ranging from C16 to C18 should be high in potential feedstock for suitable biodiesel production [58]. It is suggested that FAME with a high percentage of monounsaturation (C16-C18) is the most desirable compromise between cold flow and oxidative stability [59]. The W \begin{table}
\begin{tabular}{l c c c} \hline \hline
**Fatty Acids** & **W** & **R** & **RS** \\ \hline Saturated fatty acids (SFA) & 26.34 & 24.86 & 26.25 \\ Monounsaturated fatty acids (MUFA) & 31.80 & 31.85 & 28.91 \\ Polyunsaturated fatty acids (PUFA) & 41.86 & 43.29 & 44.83 \\ Omega-3/Omega-6 & 6.46 & 5.68 & 5.77 \\ \hline Total & 100 & 100 & 100 \\ \hline \hline \end{tabular}
\end{table}