## 4 Discussion  
The lipid fractions of algal biomass can differ significantly between species and vary under different growth conditions [27]. Furthermore, microalgae produce a variety of other lipophilic biomolecules such as pigments, waxes, proteins, and sterols. In order for lipid extraction and quantification methods to be accurate, procedures must effectively discern fatty acids from other lipid-soluble compounds, which cannot be converted to FAME. Oftentimes, lipophilic components of the biomass are co-extracted during the extraction process and can potentially overestimate the lipid yield. In particular, the polarity of solvents has a large effect on the lipid extraction yields using the conventional two-step approach. Relative to the conventional two-step method of lipid extraction and transesterification, one-step in situ transesterification offers a reliable and accurate method to measure FAME yields. Only small quantities of microalgal biomass are required for one-step in  
Figure 5: (**a**) Extraction yield based on dry biomass; FAME yield based on total extract (%DW); (**b**) FAME yield of dry biomass (%DW). situ transesterification, which provides a fast and simple method for the selection of microalgae by calculating FAME content. In the present study, the gravimetric yields of crude lipid extracts using methanol and chloroform were generally higher than the petroleum ether and ethyl ether solvent system with corresponding FAME yields of 24.11% and 12.93%, respectively. Indeed, algal lipid yields typically increase with the polarity of the solvent, which is in accordance with the literature [28]. Since total lipid yields were higher in methanol and chloroform, this polar solvent system likely co-extracted carbohydrates, protein, salts, and other polar impurities. Polar solvents can extract different lipids classes, as well as caroten, sterols, etc. [29]. Carbohydrates and proteins can account for a significant portion of microalgal biomass--as high as 24%-33% carbohydrates and 36%-44% protein in _Nannochloropsis gaditana_[30], 28% starch, and 35% proteins in _Dunaliella salina_[31]. Fatty acids are an important class of lipids with commercially viable applications using triacylglycerols as biofuels and polyunsaturated fatty acids as nutrition oils. Currently, the principal source of PUFAs is fish. However, the high demand for consumption of PUFAs is likely to not be met by fish alone. In addition, the consumption of fish oil has a number of limitations due to the potential danger of contamination and fish odor [32; 33]. Microalgae, such as _Phaeodactylum_, _Thalassioira_, and _Nannochloropsis_ species could serve as alternative sources of valuable EPA [3; 7]. Developments in cost-effective and large-scale production of microalgae would make this process more sustainable [34]. For isolating EPA from _Nannochloropsis gaditana_, numerous previous studies have focused on the conventional two-step extraction method. [PERSON] et al. used this conventional method to obtain FAME from _Nannochloropsis gaditana_ and observed that the EPA content was between 19.1% and 37.8% of the total fatty acids depending on the salinity [35]. [PERSON] et al. stated that the FAME yield under normal white light was 11.4% of dry biomass using the conventional two-step method, and the maximal EPA content was 20.3% of the total FAME from _Nannochloropsis gaditana_[36]. In the current study, the novel in situ transesterification method was optimized based on a variety of parameters. Compared to the conventional method, larger amounts of EPA were obtained in the lipid extract using this optimized method, which also demonstrates that this strain of _Nannochloropsis gaditana_ is a promising source of EPA. The fast and simple one-step method may indeed reduce the chances of EPA being oxidized, since EPA is air and light sensitive.