## 3 Results
### Biochemical Composition
#### 3.2.2 Proteins, Carbohydrates and Lipids  
As expected, with both salts, a clear tendency was observed towards an increase in protein content as a consequence of an increase in nitrogen concentration (\(p\) < 0.05). However, this difference was not sustained throughout the experiment. This result is common in batch cultures without nitrogen replenishment. As the microalgae grew, moderate-to-strong nitrogen depletion decreased amino acid synthesis, as was expected. For instance, at the onset of the experiment (day 3), a significantly high protein concentration (\(-\)390 mg mg\({}^{-1}\)) was observed in the 890 mM-treated NH\({}_{4}\)Cl and NaNO\({}_{3}\), as well as in the 445 mM-treated NaNO\({}_{3}\). As the experiment progressed, protein concentration decreased, reaching concentrations of <220 mg\({}^{-1}\) by day 18. A high decrease, from 47 to 57%, in protein content was observed in very low (N1 and C1), moderately high (N3 and C3) and high (N4 and C4) nitrogen salt concentrations with time. Moderately low salt concentrations (C2 and N2) initially yielded lower protein concentrations but showed less variation with time. In this case, the decline in protein concentration during the experiment was 39% for NaNO\({}_{3}\) and 31% for NH\({}_{4}\)Cl (Tables 3 and 4). The carbohydrate and lipid contents were influenced by the interaction between nitrogen concentration and culture age (\(p\) < 0.005) and not by the nitrogen source. The carbohydrate concentration was the least consistent in time and nitrogen concentration. For instance, during most of the experiment, carbohydrates showed higher concentrations with high NH\({}_{4}\)Cl concentrations (C3 and C4) but no clear pattern was observed with time. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline  & \multicolumn{5}{c}{**Time (Days)**} \\ \hline  & **NH\({}_{4}\)Cl μm** & **3** & **9** & **15** & **18** \\ \hline  & 111.25 & 126.5 \(\pm\) 10.5 & 72.21 \(\pm\) 20.9 & 124.4 \(\pm\) 16.6 & 102.9 \(\pm\) 11.8 \\ Protein & 222.50 & 143.4 \(\pm\) 22.8 & 146.8 \(\pm\) 11.8 & 165.8 \(\pm\) 5.8 & 128.5 \(\pm\) 23.3 \\ (\(\mathrm{\SIUnitSymbolMicro g}\) mg\({}^{-1}\)) & 445 & 230.2 \(\pm\) 16.0 & 238.3 \(\pm\) 52.1 & 171.2 \(\pm\) 43.7 & 183.8 \(\pm\) 56.7 \\  & 890 & 393.1 \(\pm\) 68.5 & 242.3 \(\pm\) 12.5 & 218.7 \(\pm\) 6.9 & 199.4 \(\pm\) 11.5 \\ \hline  & 111.25 & 350.9 \(\pm\) 45.9 & 354.3 \(\pm\) 22.2 & 279.9 \(\pm\) 45.6 & 279.1 \(\pm\) 22.9 \\ Carbohydrates & 222.50 & 351.7 \(\pm\) 106.9 & 385.6 \(\pm\) 13.6 & 380.9 \(\pm\) 29.1 & 266.6 \(\pm\) 21.4 \\ (\(\mathrm{\SIUnitSymbolMicro g}\) mg\({}^{-1}\)) & 445 & 205.1 \(\pm\) 27.7 & 348.6 \(\pm\) 20.3 & 284.2 \(\pm\) 20.6 & 248.1 \(\pm\) 16.9 \\  & 890 & 142.3 \(\pm\) 50.6 & 353.0 \(\pm\) 54.3 & 271.6 \(\pm\) 34.8 & 267.9 \(\pm\) 30.0 \\ \hline  & 111.25 & 378.6 \(\pm\) 10.5 & 497.7 \(\pm\) 11.1 & 404.7 \(\pm\) 17.7 & 387.6 \(\pm\) 11.1 \\ Lipids & 222.50 & 48.6 \(\pm\) 28.3 & 450.9 \(\pm\) 28.2 & 404.8 \(\pm\) 0.08 & 425 \(\pm\) 14.4 \\ (\(\mathrm{\SIUnitSymbolMicro g}\) mg\({}^{-1}\)) & 445 & 263.1 \(\pm\) 10.8 & 328.6 \(\pm\) 24.3 & 414.2 \(\pm\) 12.0 & 309.4 \(\pm\) 7.2 \\  & 890 & 235.0 \(\pm\) 9.4 & 319.8 \(\pm\) 6.54 & 318.8 \(\pm\) 9.0 & 309.2 \(\pm\) 14.9 \\ \hline \hline \end{tabular}
\end{table} 
Table 4: Total protein, carbohydrate and lipid contents (mean value and standard deviation) of _Thalassioin Weissflogii_ dry biomass at different days cultured at four different nitrogen concentrations of NH\({}_{4}\)Cl. By contrast, the NaNO\({}_{3}\) cultures increased their carbohydrate concentration with time and increased, on most days, with low nitrogen concentration (Table 3). As expected, the lipids reached highest concentrations at lower nitrogen concentrations. In the case of NaNO\({}_{3}\), they reached a plateau of \(\sim\)460 \(\upmu\)g mg\({}^{-1}\) for N2 and 495 \(\upmu\)g mg\({}^{-1}\) for N1 after day 6. These values were not significantly different from those observed at day 9 or 12, suggesting a relatively long stationary phase in terms of lipid yield.