## 4 Discussion  
### Microalgae Growth and Biomass Production  
As the nitrogen concentration increased in the cultures, the _T. weissflogii_ biomass concentration and cell density increased. A decrease in algal biomass concentration in low nitrate concentration was also observed [35] in _Nannochloropsis_ sp.  
As previously described, reduced nitrogen forms as ammonium or urea are preferably used for microalgae, since nitrate must be transformed into ammonium before it is utilized assimilated by algal cells, so more energy expenditure is necessary [36; 37]. This assertion remains true for lower nitrogen salt concentrations and was particularly evident for moderately low concentrations (N2 and C2), where the NH\({}_{4}\)Cl yielded a biomass peak twice as large as that yielded by the NaNO\({}_{3}\). However, high NH\({}_{4}\)Cl concentration cultures reached  
\begin{table}
\begin{tabular}{c c c c c c c c} \hline  & \multicolumn{4}{c}{**NaNO\({}_{3}\) (\(\upmu\)M)**} & \multicolumn{4}{c}{**NH\({}_{4}\)Cl (\(\upmu\)M)**} \\
**PUFA** & **111.25** & **222.5** & **445** & **890** & **111.25** & **222.5** & **445** & **890** \\ \hline
18:2 & \(1.20\pm 0.53\) & \(1.59\pm 0.46\) & \(1.32\pm 0.31\) & \(2.96\pm 0.83\) & \(0.63\pm 0.21\) & \(0.97\pm 0.38\) & \(1.38\pm 0.68\) & \(1.52\pm 0.37\) \\
18:3 & \(3.03\pm 1.13\) & \(2.82\pm 1.17\) & \(2.58\pm 1.91\) & \(4.08\pm 2.69\) & \(1.33\pm 0.29\) & \(2.57\pm 1.99\) & \(3.53\pm 1.29\) & \(4.08\pm 2.69\) \\
18:4 & \(3.11\pm 1.05\) & \(4.86\pm 1.82\) & \(3.53\pm 2.13\) & \(5.95\pm 4.67\) & \(3.61\pm 1.43\) & \(3.14\pm 1.90\) & \(5.83\pm 3.61\) & \(1.89\pm 1.45\) \\
20:4 & \(0.014\pm 0.04\) & \(0.026\pm 0.01\) & \(0.059\pm 0.04\) & \(0.043\pm 0.02\) & \(0.01\pm 0.000\) & \(0.01\pm 0.006\) & \(0.01\pm 0.007\) & \(0.02\pm 0.009\) \\
20:5n3 (EPA) & \(16.67\pm 4.35\) & \(7.58\pm 2.48\) & \(14.12\pm 3.09\) & \(7.29\pm 1.14\) & \(11.94\pm 9.47\) & \(16.99\pm 4.73\) & \(10.24\pm 3.36\) & \(11.04\pm 6.67\) \\
22:6n3 (DHA) & \(7.22\pm 1.45\) & \(4.37\pm 3.02\) & \(6.45\pm 2.76\) & \(9.24\pm 3.69\) & \(4.82\pm 2.06\) & \(8.38\pm 2.01\) & \(5.25\pm 2.44\) & \(6.77\pm 4.16\) \\ \hline \end{tabular}
\end{table}
Table 5: Polyunsaturated fatty acid (PUFA), eicosapentaenoic acid (EPA) and docosahexaenoicenoic acid (DHA) composition of _Thalass  
peak biomass three days later than similar NaNO\({}_{3}\) cultures, which suggests that at high salt concentrations, NaNO\({}_{3}\) can be assimilated slightly faster than NH\({}_{4}\)Cl.  
Considering only the cost per metric ton of each salt ($200-$430 USD ton\({}^{-1}\) NaNO\({}_{3}\) vs. $90-$200 USD metric ton\({}^{-1}\) NH\({}_{4}\)Cl) [38; 39] and the amount required to run the experiment, an estimated production (mg USD\({}^{-1}\)) was computed. Since NH\({}_{4}\)Cl is less than half the price of NaNO\({}_{3}\), treatments with low NH\({}_{4}\)Cl were the most cost-effective in all cases. All the treatments reached their most cost-effective values after 15 or 18 days. However, differences in time and growth rate were also important for estimated costs. For instance, C1 and C2 (low and moderately low NH\({}_{4}\)Cl) provided a maximum of >6 mg DW USD\({}^{-1}\) after 15 days, whereas C3 and C4 (moderately high and high NH\({}_{4}\)Cl) provided a maximum of 3-4 mg DW USD\({}^{-1}\) after 15 and 18 days, respectively. By contrast, N1 reached a maximum of 3.2 mg DW USD\({}^{-1}\) after 18 days, and N2-N4 provided only 1.44-1.68 mg DW USD\({}^{-1}\) after 15 or 18 days.