## 4 Discussion  
The optimization of the culture medium is one of the most important parameters to obtain concentrated cultures with high DHA amount of _Aurantiochytrium_ sp., and the lab-scale optimization carried out in this work allowed significant improvements that can be key to industrial cultivation. _Aurantiochytrium_ sp. was first cultivated in MB10, as it is the stock medium used in-house for heterotrophic cultivation of other microorganisms. Nevertheless, in this strain, the preliminary growth using this medium was unsatisfactory, given its low growth rates and biomass productivity. As MB10 contains NaCl, the presence of chloride ions in the medium was detected. This is undesired, because such ions are corrosive, affecting the operation of industrial fermentation vessels. Thus, to prevent \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Medium** & **Proteins (\%)** & **Lipids (\%)** & **Carbohydrates (\%)** & **Ashes (\%)** \\ \hline MB10 & 23.57 \(\pm\) 0.65 \({}^{\text{a}}\) & 22.78 \(\pm\) 0.20 \({}^{\text{a}}\) & 28.13 \(\pm\) 0.69 \({}^{\text{a}}\) & 25.52 \(\pm\) 2.46 \({}^{\text{a}}\) \\ IOM & 14.02 \(\pm\) 0.04 \({}^{\text{c}}\) & 14.74 \(\pm\) 0.15 \({}^{\text{c}}\) & 59.05 \(\pm\) 3.47 \({}^{\text{b}}\) & 12.19 \(\pm\) 0.05 \({}^{\text{b}}\) \\ WOM & 20.55 \(\pm\) 0.33 \({}^{\text{b}}\) & 31.14 \(\pm\) 3.41 \({}^{\text{b}}\) & 32.99 \(\pm\) 2.71 \({}^{\text{a}}\) & 15.31 \(\pm\) 0.36 \({}^{\text{b}}\) \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Proximate composition of _Aurantiochytrium_ sp. AF0043 biomass strain cultures, grown in MB10 medium (control), inorganic optimized medium (IOM), and waste-based optimized medium (WOM). Values are the mean and corresponding standard deviation of three biological replicates. Different letters indicate significant differences between biomass grown in different media. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**FAME** & **MB10** & **IOM** & **WOM** \\ \hline C14:0 & 14.53 \(\pm\) 0.52 & 8.22 \(\pm\) 4.14 & 9.40 \(\pm\) 0.49 \\ C15:0 & 0.58 \(\pm\) 0.05 & 1.71 \(\pm\) 1.71 & 2.96 \(\pm\) 0.18 \\ C16:1 & 2.73 \(\pm\) 1.07 & 2.20 \(\pm\) 0.91 & 3.12 \(\pm\) 0.10 \\ C16:0 & 72.26 \(\pm\) 0.27 & 64.21 \(\pm\) 8.31 & 46.32 \(\pm\) 1.22 \\ C17:0 & 0.04 \(\pm\) 0.04 & 0.33 \(\pm\) 0.33 & 1.16 \(\pm\) 0.14 \\ C18:0 & 0.24 \(\pm\) 0.20 & 0.55 \(\pm\) 0.55 & 1.68 \(\pm\) 0.58 \\ C22:5 (DPA) & 0.19 \(\pm\) 0.11 & 5.74 \(\pm\) 3.93 & 5.98 \(\pm\) 5.98 \\ C22:6n-3 (DHA) & 10.12 \(\pm\) 2.13 & 17.05 \(\pm\) 11.10 & 29.66 \(\pm\) 3.28 \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Fatty acid methyl esters profile of _Aurantiochytrium_ sp. AF0043 cultures, grown in MB10 medium (control), inorganic optimized medium (IOM), and waste-based optimized medium (WOM). Values are given as the mean \(\pm\) standard deviation percentage of the total FAME detected. this, the inclusion of Cl must be avoided in the industrial medium formulation [30; 34]. However, most thraustochytrids require sea salts for growing, which are essential for lipid synthesis and osmotic adjustment [26; 30; 34]. Therefore, to adjust the medium to the species under study and bearing in mind the aim of maintaining its industrial feasibility, an extensive literature review was performed, focusing on the macronutrients, trace metals, vitamins and N sources of the culture medium. This search resulted in the MT1 medium, which was formulated taking into account the best growth performance and DHA accumulation. In MT1, Na\({}_{2}\)SO\({}_{4}\) is the largest contributor to the medium salinity, eliminating chlorine ions from the medium without compromising the organism's growth or DHA production. In fact, [PERSON] et al. [30] found out that the replacement of sea salt (2% w/v) by Na\({}_{2}\)SO\({}_{4}\) (1% w/v) improved the maximum concentration of biomass from 35 to around 39 g/L, while DHA yield (7.95 g/L) increased from 40% to 44% of the total lipids in the thraustochytrid _Schizochytrium_ sp., corroborating the feasibility of NaCl replacement.