## 4 Discussion  
Effect of the Replacement of Organic Nitrogen Sources with Pre-Treated Effluent Enriched with Inorganic Nitrogen on Biomass and Lipid Productivity  
Nitrogen serves as an essential component for eukaryotic microalgae cells for the synthesis of important cellular constitutes (peptides, enzymes, ATP and DNA) in order to sustain growth and development [60]. Yeast extract is an organic nitrogen source widely used in microbial fermentation due to its essential content in amino acids, vitamins and trace elements [61]. It is the most expensive ingredient in thraustochytrids culture media, considering that in order to reach a biomass productivity of 60 g L\({}^{-1}\) and DHA content of 20 g L\({}^{-1}\), the addition of 4% _w/v_ yeast extract is necessary [36; 49]. Furthermore, nitrogen sources, which favor optimal growth and development, are species-specific [35]. Therefore, several nutrient sources, organic or none, have been proposed in order to reduce production cost and optimize nutrient media for optimal biomass productivity and DHA yield of _S. limacinum_. According to [PERSON], et al. [62], the highest biomass productivity of 15 g L\({}^{-1}\) was achieved when the culture media was supplied with yeast extract and corn steep liquor in batch cultures with regard the organic nitrogen sources. In the same study, when inorganic nutrient sources were used, the biomass increased approximately 40% with ammonium sulfate, urea and ammonium acetate. However, ammonium acetate was more favorable for lipid accumulation and DHA yield. [PERSON], et al. [63] utilized pre-treated effluent as an alternative nutrient source for heterotrophic cultivation of _S. limacinum_ with pure glycerol. Even though nutrient removal of ammonia and phosphates was successful, which was the main focus of the study, DHA content was not measured, and their low biomass productivity (1.3 g L\({}^{-1}\)) constitutes a limiting factor for industrial production standards. In this current study, organic nutrients sources (yeast extract, glycerol and peptone) were successfully substituted with low cost nitrogen sources (NH\({}_{4}\)Cl), crude glycerol and effluent digestate rich in trace elements, achieving a biomass productivity 49.2 \(\pm\) 2.7 g L\({}^{-1}\), three times higher compared to the treatment with the organic nitrogen sources and comparable to earlier mentioned studies, while the total lipid production was equal to that obtained with organic sources. This is in accordance with [PERSON] et al. [40], where organic nitrogen inhibited cell growth because it was utilized for the synthesis of cell components rather than cell reproduction [64], which could explain the significant lower biomass recorded in the treatment with yeast extract and peptone. In the same study, when monosodium glutamate was used as the inorganic nitrogen source, lipid accumulation was enhanced. One of the essential amino acids contained in abundance in yeast extract is glutamic acid (glutamate) [65]. It has \begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**Effluent Concentration** & \begin{tabular}{c} **Myristic Acid** \\ **14:0** \\ **(\%)** \\ \end{tabular} & \begin{tabular}{c} **Palmitic Acid** \\ **16:0** \\ **(\%)** \\ \end{tabular} & \begin{tabular}{c} **Stearic Acid** \\ **18:0** \\ **(\%)** \\ \end{tabular} & \
\begin{tabular}{c} **DPA** \\ **22:5** **n-6** \\ **(\%)** \\ \end{tabular} &
\begin{tabular}{c} **DHA** \\ **22:6** **n-3** \\ **(\%)** \\ \end{tabular} \\ \hline
0 & 0.88 \(\pm\) 0.9  & 54.70 \(\pm\) 17.8  & 40.79 \(\pm\) 16.9  & n.d & 3.33 \(\pm\) 1.7  \\
8 & 0.93 \(\pm\) 0.2  & 17.72 \(\pm\)1.3  & 80.79 \(\pm\) 1.5  & n.d & 0.56 \(\pm\) 0.1  \\
16 & 0.44 \(\pm\) 0.4  & n.d & n.d \\
32 & 0.73 \(\pm\) 0.7  & 20.10 \(\pm\) 0.8  & 74.83 \(\pm\) 0.8  & n.d & 7.5\(\pm\) 1.2  \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Fatty acid (percent of total fatty acids) composition of dried algal biomass of _S.