**3. Valorization of three algal species**

Three different microalgae species were isolated from the three level of salinity of Sfax solar saltern: the diatom *Halamphora* sp. (45–80 PSU) cyanobacterium *Phormidium versicolor* (80–200 PSU) and the chlorophyceae *Dunaliella salina* (200–450 PSU) (**Figures 1** and **3**). These species were cultured in the laboratory. The extraction of the various pigments and biomolecules contained in these species was carried out.

#### **3.1 Isolation and culture conditions**

*Halamphora* sp. was isolated via micromanipulation and serial dilution from a collected water sample. Both *D. salina* and *P. versicolor* were isolated on agar medium. Several antibiotic and antifungal treatments were performed in order to obtain monoclonal and axenic cultures. These three algal species were batch cultured in flasks 500 ml Erlenmeyer flask with artificial sea water of 80 PSU. The cultures were initiated with cell densities of 10<sup>6</sup> cells ml−1 for *D. salina* [31], 50,000 cells ml−1 for *Halamphora* sp. [19] and an initial concentration of chlorophyll *a* of 0.005 μg ml−1 for *P. versicolor* because the numeration was impossible (the filaments intertwine) [9]. *P. versicolor* was cultivated using BG-11 medium as culture medium [32], *D. salina* was grown in Walne's growth medium as modified by Guermazi, Elloumi, Ayadi, Bouain and Aleya [17] and for *Halamphora* sp., the culture was carried out in F/2 Provasoli medium [33]. Cultures were maintained in incubator (FRIOCELL) and incubated in 24-light cycles at (25°C). *Phormidium* and *Halamphora* were reared under light intensity of 130 μmol photons m−2 s−1, while Dunalielal was cultured under low irradiance of 27 μmol photons m−2 s−1.

**Figure 3.**

*Microscopic observation (G ×100) of (a) P. versicolor, (b) D.salina and (c) Halamphora sp. isolated from the Sfax solar salternand cultured in the laboratory.*

The biomass of each microalga was separated from the culture media by centrifugation (4500 × g, 10 min), and the pellet was washed with distilled water and centrifuged again at 4500 × g for 10 min (the washing was repeated twice). The pellet was freeze-dried and stored at −70°C.

## **3.2 Growth tracking**

The growth of *D. salina* and *Halamphora* sp. was determined by a daily cell count using a Malassez hemocytometer under a light microscope.

The cyanobacterium *P. versicolor* being filamentous, the chlorophyll *a* was determined to assess the growth of this cyanobacterium according to the equation of Speziale, Schreiner, Giammatteo and Schindler [34]:

$$\begin{array}{c} \text{Chl } a \text{(}\mu\text{g l}^{-1}\text{)}=\text{[}11.4\text{7 (OD}\_{664\text{nm}}\text{ - OD}\_{750\text{nm}}\text{)}\\ \text{-0.4 (OD}\_{664\text{nm}}\text{ - OD}\_{750\text{nm}}\text{)} \,\text{l}\,\nu/\text{ V} \end{array}$$

with. OD: Optical density. υ: volume of the acetone extract (ml). V: volume of the algae suspension (ml).
