# Antioxidant Production in _Dunaliella_
## 3 Cultivation Conditions  
In addition to carbon, light energy, and water, _Dunaliella_ requires certain mineral nutrients for growth. A suitable medium can be prepared from natural or artificial seawater enriched with nutrients (carbon, nitrogen, phosphorous, sulphur, iron, and magnesium), trace metals, and vitamins [40]. Carbon dioxide (CO2), inorganic carbon (e.g., NaHCO3, Na2 CO3), and organic carbon (e.g., sodium acetate, glucose, glycerol) can be used as carbon sources. Nitrate, ammonia, and urea are commonly used as nitrogen sources for the synthesis of amino acids, nucleotides, chlorophylls and phycobilins [103]. Phosphorous is needed for several metabolic processes (ATP, DNA, RNA, and phospholipids). Iron acts as a cofactor for many enzymes (e.g., ferredoxins, catalases, nitrogenases, nitrates), and sulphur is needed for the biosynthesis of specific amino acids (cysteine, methionine). Magnesium is required for t chlorophyll biosynthesis, and other trace minerals serve as cofactors for various enzymes. _Dunaliella_ needs light and temperature to assimilate carbon during photosynthesis and enhance biomass productivity and growth [104]. Growth conditions can be classified into three types based on energy and carbon sources (Table 3). \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Characteristics** & **Photo-Autotrophic** & **Heterotrophic** & **Mixotrophic** \\ \hline Carbon assimilation process & Photosynthesis & Aerobic respiration & Photosynthesis and aerobic respiration \\ \hline Energy sources & Light (solar or artificial) & Organic carbon (e.g., glucose, acetic acid glycerol, wastewaters) & Light and organic carbon \\ \hline Carbon sources & Inorganic carbon (CO2, NaHCO3) & Organic carbon & Inorganic and organic \\ \hline Light energy & Mandatory & Not required & Not essential \\ \hline Photo-inhibition effect & High & No effect & Low \\ \hline Growth-limiting factor & Light & Oxygen & Light and oxygen \\ \hline Photo-oxidative damage risk & High & Very low & Protected by accumulating oxygen \\ \hline Types of bioreactors & Photo-bioreactors and open ponds & Fermenters & Photo-bioreactors, open ponds \\ \hline Availability of bioreactor’s & Photo-bioreactor & Commercially available & Bioreactor \\ vessels & & & \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Characteristics of microalgae growth modes [105; 106]. Photoautotrophic culturing is the most common strategy for growing _Dunaliella_ biomass. However, in large cultures, cell-shading becomes an issue, limiting light penetration into the culture, leading to lower amounts of biomass [107]. In mixotrophic cultivation, the microalgae use CO\({}_{2}\) and organic carbon (acetate, glucose) simultaneously along with light energy; respiratory and photosynthetic metabolism operates concurrently [105]. This increases biomass sproduction and results in a higher lipid production compared to that observed in photoautotrophic cultures [107; 108; 109; 110; 111; 112; 113; 114; 115; 116; 117; 118], though at increased cultivation costs. Although heterotrophic cultivation of algae [106] eliminates the requirements for light and facilitates biomass production in the dark, attempts to grow _D. para_ heterotrophically were unsuccessful [110]. However, some _Dunaliella_ strains can grow heterotrophically using organic carbon (such as tryptone, yeast extract, and urea, etc.) as an energy source, together with nutrients [128; 129; 130; 131; 132; 133]. No studies have been published supporting the heterotrophic growth of _Dunaliella_ strains at a commercial scale. _Dunaliella_ can grow phototrophically at temperatures ranging between 10 \({}^{\circ}\)C and 30 \({}^{\circ}\)C and in a wide variety of media, where the maximum cells density range between 0.3 \(\times\) 10\({}^{6}\) cells mL\({}^{-1}\) and 24 \(\times\) 10\({}^{6}\) cells mL\({}^{-1}\)[25]. In addition, these species are halotolerant and can grow in a high saline medium (0.5-4.0 M NaCl) [111].