# Enhancement of Metabolite Production in High-Altitude Microalgal Strains by Optimized C/N/P Ratio
## 2 Materials and Methods
### Quantification of Biomass and Metabolites  
The biomass produced was concentrated using a lab-scale electrofoltation device [104], washed thrice with distilled water, reconcentrated by centrifugation (2876\(\times\) g, 20 \({}^{\circ}\)C, 20 min), lyophilized, and stored (4 \({}^{\circ}\)C) until use. Finally, the different components of the strains were measured in triplicate.  
For carbohydrates, 5 mg of grounded biomass were mixed with 5 mL of 1 M H\({}_{2}\)SO\({}_{4}\) in acid-resistant test tubes, vortexed (Multi Reax, Heidoplh, Schwabach, Germany) at 1500 rpm for 10 min, and incubated (100 \({}^{\circ}\)C, 60 min). The mixture was cooled down at room temperature (30 min) and centrifuged (2876\(\times\) g, 20 min). Two milliliters of the supernatant was mixed with 1 mL of phenol solution (5% \(w/v\)) and 5 mL of concentrated H\({}_{2}\)SO\({}_{4}\) and cooled down at room temperature (30 min). The sample was measured at 485 nm, and the final content was calculated in % \(w/w\). [103].  
\begin{table}
\begin{tabular}{c c c c} \hline
**Factor** & **Units** & **Low Level (\(-\)1)** & **High Level (\(+\)1)** \\ \hline Carbon source & g/L & 0.2 & 0.5 \\ NaNO\({}_{3}\) & g/L & 0.125 & 0.25 \\ K\({}_{2}\)HPO\({}_{4}\) & g/L & 0.0375 & 0.075 \\ KH\({}_{2}\)PO\({}_{4}\) & g/L & 0.0875 & 0.175 \\ \hline \end{tabular}
\end{table}
Table 1: Variables evaluated with their respective levels.  
The total lipids were measured using the method described by [PERSON] et al. [105]. Briefly, 5 mg of ground biomass were mixed with 2 mL of concentrated H\({}_{2}\)SO\({}_{4}\) in acid-resistant test tubes and vortexed (Multi Reax, Heidolh) at 1500 rpm for 5 min. The sample was then incubated (100 \({}^{\circ}\)C, 10 min) and cooled down in an ice bath for 5 min. Five milliliters of fresh phospho-vanillin reagent was added to the sample and incubated for a second time (37 \({}^{\circ}\)C, 15 min). The final sample was centrifuged (2876\(\times\) \(g\), 20 min), the supernatant was measured at 530 nm, and the final content was calculated in % \(w/w\).  
The total content of proteins was measured using the method described by [PERSON] et al. [106]. Briefly, 5 mg of grounded biomass was mixed with 0.5 mL of distilled water and 0.5 mL of 10% \(w/v\) of SDS solution and vortexed (Multi Reax, Heidolph) at 1500 rpm for 10 min. The sample was centrifuged (8000\(\times\) \(g\), 5 min), and 1 mL of the supernatant was diluted in 5 mL of reagent C using the Lowry method. Finally, 0.5 mL of Folin reagent (diluted 3 times) was added to the mixture and incubated at room temperature for 30 min. The sample was measured at 750 nm, and the final content was calculated in % \(w/w\).  
Finally, the content of total carotenoids was measured according to [PERSON] et al. [107]. Briefly, 100 mg of grounded biomass was mixed with 1 mL of acetone and 10 mg of MgSO\({}_{3}\). The mixture was vortexed (Multi Reax, Heidolph) at 2000 rpm for 10 min. The sample was then centrifuged (8000\(\times\) \(g\), 20 min). The supernatant was diluted 4 times in acetone and measured at 470 nm, and the final content was calculated in % \(w/w\).