# Enhancement of Metabolite Production in High-Altitude Microalgal Strains by Optimized C/N/P Ratio
## 3 Results  
### Production of Carbohydrates under Different C/N/P  
The experimental data obtained from the C/N/P ratio on the production of carbohydrates were fitted on two models (linear (L) and quadratic (Q) with a _p_-value of 0.05 (_p_ = 0.05)). The statistical software provided a number to identify them; in this case NaNO\({}_{3}\) was tagged with the number 1, followed by the phosphate buffer and carbon source: (NaNO\({}_{3}\) (1), K\({}_{2}\)HPO\({}_{4}\) + KH\({}_{2}\)PO\({}_{4}\) (2), and carbon source (3). This tag is used by the software to identify possible interactions between two or more variables (example: 1L_by_2L represents and interaction between the lineal model of NaNO\({}_{3}\) and K\({}_{2}\)HPO\({}_{4}\) + KH\({}_{2}\)PO\({}_{4}\)). In the Y-axis of each Pareto chart can be found the analysis for every single factor adjusted by the two models (Q and L) with the numbers of the three variables evaluated (1, 2 or 3), and on the right side of each row, the assigned number allows us to understand which level has  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multirow{2}{*}{**Standard Run**} & \multirow{2}{*}{**Block**} & \multirow{2}{*}{**NaNO\({}_{3}\)**} & \multirow{2}{*}{**K\({}_{2}\)HPO\({}_{4}\)**} & \multirow{2}{*}{**KH\({}_{2}\)PO\({}_{4}\)**} & \multirow{2}{*}{**Carbon Content**} \\ \cline{3-3} \cline{5-6}  & & **g/L** & & **g/L** & **g/L** & **(g/L)** \\ \hline
1 & & 0.125 & 0.0375 & 0.0875 & 0.20 \\
2 & & 0.125 & 0.075 & 0.175 & 0.50 \\
3 & 1 & 0.25 & 0.0375 & 0.0875 & 0.50 \\
4 & & 0.25 & 0.075 & 0.175 & 0.20 \\
5 (Central point) & & 0.1875 & 0.056 & 0.131 & 0.35 \\ \hline
6 & & 0.125 & 0.0375 & 0.0875 & 0.50 \\
7 & & 0.125 & 0.075 & 0.175 & 0.20 \\
8 & 2 & 0.25 & 0.0375 & 0.0875 & 0.20 \\
9 & & 0.25 & 0.075 & 0.175 & 0.50 \\
10 (Central point) & & 0.1875 & 0.056 & 0.131 & 0.35 \\ \hline
11 & & 0.083 & 0.056 & 0.131 & 0.35 \\
12 & & 0.292 & 0.056 & 0.131 & 0.35 \\
13 & & 0.1875 & 0.0249 & 0.0581 & 0.35 \\
14 & 3 & 0.1875 & 0.0876 & 0.2044 & 0.35 \\
15 & & 0.1875 & 0.0 the most effect. Lower numbers (-) indicate that lower levels of that specific variable are positively or negatively affecting the response.  
According to the Pareto chart (Figure 1a), when sodium carbonate (Sc) is used as a carbon source, the carbohydrate synthesis is significantly affected by all three factors of the C/N/P ratio (\(p\) = 0.05). The surface response (Figure 1b) shows that high concentrations of NaNO\({}_{3}\) (0.15-0.25 g/L) and high concentrations of the carbon source (2.64-4.41 g/L of Na\({}_{2}\)CO\({}_{3}\)) positively affect the synthesis of carbohydrates up to 50% \(w/w\) of the total biomass. In the case when sodium bicarbonate (Sb) is employed, the phosphate buffer and the carbon source significantly affect the synthesis of carbohydrates (Figure 1c). The surface response (Figure 1d) shows that high concentrations of K\({}_{2}\)HPO\({}_{4}\) and KH\({}_{2}\)PO\({}_{4}\) (0.075 and 0.175 g/L, respectively), and high concentrations of the carbon source (2.1-3.49 g/L of NaHCO\({}_{3}\)) positively affect the synthesis of carbohydrates up to 60% (\(w/w\)) of the total biomass. Finally, the Pareto chart of sodium acetate (Sa) (Figure 1e) shows that none of the analyzed nutrients in the designated concentrations significantly affects (\(p\) = 0.05) the synthesis of carbohydrates; however, the surface response (Figure 1f) shows that high concentrations of K\({}_{2}\)HPO\({}_{4}\) and KH\({}_{2}\)PO\({}_{4}\) (0.075 and 0.175 g/L, respectively), and high concentrations of the carbon source (0.85-1.36 of C\({}_{2}\)H\({}_{3}\)NaO\({}_{2}\)) can increase the concentration of carbohydrates up to 45% (\(w/w\)) of the total biomass in _Chlorella_ sp. UFFS019.  
Figure 1: Pareto charts and surface response of carbohydrate production using Na\({}_{2}\)CO\({}_{3}\) (**a**,**b**), NaHCO\({}_{3}\) (**c**,**d**), and C\({}_{2}\)H\({}_{3}\)NaO\({}_{2}\) (**e**,**f**).