# Enhancement of Metabolite Production in High-Altitude Microalgal Strains by Optimized C/N/P Ratio
## 4 Discussion  
The optimization of culture media is considered one of the key processes to improve the production of biomass and target metabolites, whereby it is possible to adjust the concentrations of certain nutrients (both macro and micronutrients) depending on the strain. The enhancement of the different metabolites can be manipulated through growth conditions; therefore, the main strategies include nutritional factors such as nutrient limitation/supplementation (nitrogen, phosphorus, sulfur, iron), and continuous addition of carbon source, as well as factors such as light intensity, pH, temperature, and salinity [108]. In recent years, different researchers have demonstrated the efficiency of this approach, especially using one-factor-at-a-time (OFAT) experiments, which involve the adjustment of one factor while the others are kept constant. Examples of this approach can be found in the scientific literature with a wide range of strains including (but not limited to) _Botryococcus braunii_[109], _Chlorella vulgaris_[110], _Chlorococcum_ sp. (Chlorophyta) [111], _Vischeria vischeri_ (formerly _Eustigmatos vischeri_) (Ochrophyta, Eustigmato  
Figure 5: Biomass production and consumption of NO3 and PO4 in the optimized C/N/P ratio for production of carbohydrates (**a**), proteins (**b**) and lipids (**c**). Figure 6: One-way ANOVA between the expected and obtained results to produce carbohydrates and lipids in _Chlorella_ sp. UFPS019 and proteins in _Scenadesmus_ sp. UFPS021. phyceae) [112], _Isochrysis galbanu_ (Haptophyta, Coccolithophyceaeae) [113], _Lobosphaera incisa_ (Chlorophyta) [114], and _Picochlorum soloecismus_ (Chlorophyta) [115]. However, OFAT is considered time-consuming and labor-intensive when several variables may affect the outcome. In this case, due to their intrinsic nature of combining multiple salts in different concentrations, algal culture media are a perfect candidate for more robust and advanced methodologies. In recent years, the optimization of culture media and extraction of different metabolites through the application of design of experiments (DoE) has become more common [116; 117; 118; 119; 120]. Table 5 present a list of the most recent papers where the improvement of a specific metabolite was achieved through the application of DoE. The most evaluated metabolites are lipids (including hydrocarbons) [23; 93; 97; 121; 122; 123; 124; 125; 126; 127; 128; 129]. In the second position can be found the optimization of carbohydrate synthesis [93; 131; 132], followed by the enhancement of protein synthesis [97; 98]. The latter has occurred due to the international interest in the application of algal biomass as a replacement for biofuel production, which remains a strong alternative within the international scientific community [32]. In the quest to improve the synthesis of each of the algal metabolites, the most studied components are the nitrogen and carbon sources [121; 122; 123; 124; 125; 126; 127; 128; 129]. According to [PERSON] et al. [133], nitrogen limitation causes an interruption in amino acid synthesis, and photosynthetically fixed carbon in the Calvin cycle is then converted to carbohydrates. Another nutrient of great importance is phosphate, which is essential for starch and sucrose synthesis; however, high concentrations of P inhibit the action of enzymes such as ADP-glucose phosphorylase [134].