# Enhancement of Metabolite Production in High-Altitude Microalgal Strains by Optimized C/N/P Ratio
## 3 Results
### Production of Total Carotenoids under Different C/N/P  
In the case of total carotenoids, none of the analyzed variables in any of the different carbon sources studied (Figure 4a,c,e) showed a significant effect. In the case of Na\({}_{2}\)CO\({}_{3}\) (Sa), the surface response (Figure 4b) shows that lower concentrations of the carbon source (<0.9 g/L of Na\({}_{2}\)CO\({}_{3}\)) and K\({}_{2}\)HPO\({}_{4}\), and KH\({}_{2}\)PO\({}_{4}\) (<0.0375 and 0.0875 g/L, respectively) might increase the content of total carotenoids. In the case of NaHCO\({}_{3}\) (Sb) (Figure 4d), lower content of carbon source (<0.7 g/L of NaHCO\({}_{3}\)) and high levels of K\({}_{2}\)HPO\({}_{4}\) and KH\({}_{2}\)PO\({}_{4}\) (>0.075 and 0.175 g/L, respectively) can increase the concentration of total carotenoids. Finally, when sodium acetate (Sa) is used (Figure 4f), high levels of NaNO\({}_{3}\) (>0.25 g/L) and K\({}_{2}\)HPO\({}_{4}\) and KH\({}_{2}\)PO\({}_{4}\) (>0.075 and 0.175 g/L, respectively) can increase the final content of total carotenoids in _Desmodesmus_ sp. UFPS020. By analyzing the results from the interactions in the C/N/P ratio, the values of the three variables were adjusted to increase the production of carbohydrates, proteins, and  
Figure 4: Pareto charts and surface response of total carotenoids production using Na\({}_{2}\)CO\({}_{3}\) (**a,b**), NaHCO\({}_{3}\) (**c,d**), and C\({}_{2}\)H\({}_{3}\)NaO\({}_{2}\) (**e,f**). lipids in _Chorella_ sp. UFPS019 and _Desmodesm_ sp. UFPS021. Table 3 represents the highest scenarios for increasing each metabolite in the designated strains. Both algae were grown in a 10 L flask (0.6 L/min of filtered air, 12:12 h light/dark cycle at 100 \(\upmu\)mol\(\cdot\)m\({}^{-2}\cdot\)s\({}^{-1}\)) for 20 days for carbohydrates and proteins, and 40 days for lipids. As a control, both strains were grown in [PERSON]'s basal medium without any modification. The biomass produced was concentrated by electroflocculation [104]. The culture media were filtered on 0.45 \(\upmu\)m GF-C (Sartorius, Germany) to remove excess cells, and the media were analyzed for the final content of NO\({}_{3}\) and PO\({}_{4}\) using HANNA test kits (HI 93728-01, and HI 93713-01). The experimental data for each of the metabolites were adjusted to the exponential or logarithmic phase of microalgal growth, in which the maximum specific speed (\(\upmu\)max), saturation, and affinity constants for NO\({}_{3}\) and PO\({}_{4}\) (\(K_{s}\)), as well as the yield coefficient, define the biomass production rate from a mass measure (\(Y_{\frac{X}{8}}\)). Table 4 presents the data obtained for each strain under optimal conditions. \begin{table}
\begin{tabular}{c c c} \hline \hline \multicolumn{3}{c}{_Chlorella_ sp. UFPS019 (Carbohydrates)} \\ \hline \(\upmu\) & & 0.0029 h\({}^{-1}\) \\ \(dt\) & & 120 h\({}^{-1}\) \\ \hline \multirow{4}{*}{\(Y_{\frac{X}{8}}\)} & NO\({}_{3}\) & PO\({}_{4}\) \\  & 0.2819 & 0.0698 \\ \cline{1-1}  & \(K_{s}\) & 0.1885 & 0.0768 \\ \hline \multicolumn{3}{c}{_Chlorella_ sp. UFPS019 (Lipids)} \\ \hline \(\upmu\) & & 0.00647 h\({}^{-1}\) \\ \(dt\) & & 120 h\({}^{-1}\) \\ \hline \multirow{4}{*}{\(Y_{\frac{X}{8}}\)} & NO\({}_{3}\) & PO\({}_{4}\) \\  & 0.1566 & 0.0318 \\ \cline{1-1}  & 0.004 & 0.001209 \\ \hline \multicolumn{3}{c}{_Desmodesm_ sp. UFPS021 (Proteins)} \\ \(\upmu\) & & 0.00567 h\({}^{-1}\) \\ \(dt\) & & 120 h\({}^{-1}\) \\ \hline \multirow{4}{*}{\(Y_{\frac{X}{8}}\)} & NO\({}_{3}\) & PO\({}_{4}\) \\  & 0.36 & 0.0775 \\ \cline{1-1}  & 0.0284 & 0.0772 \\ \hline \hline \end{tabular}
\end{table} 
Table 4: Growth parameters for production of carbohydrates, proteins, and lipids.