###### Abstract  
Microalgae are a valuable source of lipid feedstocks for biodiesel and valuable omega-3 fatty acids. _Nannochloropsis gaditana_ has emerged as a promising producer of eicosapentaenoic acid (EPA) due to its fast growth rate and high EPA content. In the present study, the fatty acid profile of _Nannochloropsis gaditana_ was found to be naturally high in EPA and devoid of docosahexaenoicenoic acid (DHA), thereby providing an opportunity to maximize the efficacy of EPA production. Using an optimized one-step in situ transesterification method (methanol:biomass = 90 mL/g; HCl 5% by vol. ; 70 \({}^{\circ}\)C; 1.5 h), the maximum fatty acid methyl ester (FAME) yield of _Nannochloropsis gaditana_ cultivated under rich condition was quantified as 10.04% \(\pm\) 0.08% by weight with EPA-yields as high as 4.02% \(\pm\) 0.17% based on dry biomass. The total FAME and EPA yields were 1.58- and 1.23-fold higher separately than that obtained using conventional two-step method (solvent system: methanol and chloroform). This one-step in situ method provides a fast and simple method to measure fatty acid methyl ester (FAME) yields and could serve as a promising method to generate eicosapentaenoic acid methyl ester from microalgae. EPA; FAME; one-step in situ transesterification; _Nanochloropsis gaditana_  
[PERSON] 1,2, [PERSON] 2, [PERSON] 2, [PERSON] 2 and [PERSON] 1,*  
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approximately 39.1 USD/kg dry weight, when microalgae are phototrophically cultivated in flat panel reactors with high irradiance [8]. It potentially could be reduced to 11.9 USD/kg with advances in biological productivity and improvements in the engineering parameters [8]. Among the candidate microalgal organisms for omega-3 production, the Nannochloropsis genus has emerged as a promising source of EPA [3,9]. A number of factors affecting the biomass productivity and EPA content of Nannochloropsis spp. have been reported. Application of environmental stressors to trigger high biomass accumulation and impact lipid productivity have been successfully applied by using various nitrogen sources [10], controlling the concentration of nitrogen [11], manipulating the N:P supply ratio [12], and altering salinity, pH [13], and light intensity [14]. Furthermore, genetic tools and genomic information for a number of Nannochloropsis strains are currently under development [15]. The conventional two-step approach to algal fatty acid quantification suffers from compounding inefficiencies [16] and extraction yields that are highly dependent on the polarity of the solvents. A recent report demonstrated when polar solvent (chloroform/methanol (2:1), ethanol and acetone) was used for extraction, the lipid yield was much higher than by non-polar solvent (hexane) [17]. As a result, researchers have worked toward developing a one-step in situ transesterification method especially for algal biomass. In situ transesterification means the direct conversion of the lipids in algal biomass into fatty acid methyl esters using either acid or base catalysts, or other catalysts without lipid extraction procedure [18]. In addition, in situ methods provide more reliable and accurate data than gravimetric methods, in which the weight often includes non-saponifiable lipids and overestimates the extracts [19,20]. Related studies on biodiesel production from dried or wet microalgal biomass using in situ transesterification have been summarized by [PERSON] et al. [21]. Unfortunately, much less attention has been paid to providing a simple, reliable, and rapid method to quantify the yield of omega-3 fatty acids from Nannochloropsis gaditana and other microalgae. Therefore, in the present study, a novel one-step in situ transesterification procedure to quantify EPA and other fatty acids was examined in Nannochloropsis gaditana. Reaction parameters including methanol amount, concentration of acid catalyst, reaction time, and temperature were optimized in batch reaction mode.