**3.4 Determination of dry matter, proteins, lipids, Total sugars and phenolic compounds**

The dry matter of microalgae was determined according to the AOAC standard methods [39]. The protein assay method of Lowry [40] was used by the combination of Folin with Biuret's reagents. The Lipids content was determined gravimetrically after the Soxhlet extraction of dried samples with hexane for 2 hours using Nahita Model 655 (Navarra, Spain). The sugars were estimated by phenol-sulfuric acid method [41] using glucose as a standard. The total phenol content of the *P. versicolor* extract was determined by the method of Singleton and Rossi [42].

#### **3.5 Determination of mineral content of** *Halamphora* **sp.**

The analyses of sodium, potassium, calcium, magnesium, iron, copper, and zinc contents in *Halamphora* sp. were carried out using the inductively coupled plasma optical emission spectrophotometer (ICP-OES) Model 4300 DV, PerkinElmer, Shelton, CT, USA, according to the method of AOAC 1999 [43]. Measurements were done in triplicates.

#### **3.6 Determination of fatty acids profile of** *Halamphora* **sp***.* **and** *D. salina*

For fatty acids analyses, cultures were harvested at the end of the log phase. All lipids were evaporated to dryness with nitrogen and concentrated with hexane. Fatty acids methyl esters (FAMEs) were prepared from the lipid extract by transesterification using a direct transmethylation method according to Lepage and Roy [44]. The FAMEs were then extracted with hexane and determined quantitatively by capillary gas chromatography. We used a Chromopack, CP 9001 gas chromatograph, HPS 5890 series II chromatograph, equipped with a polar 25-m capillary column CP wax 58 (Varian SA, France) (0.32 mm diameter and a layer thickness of 0.52 mm), and a flame ionization detector (FID). We used a splitless injection system with nitrogen as the carrier gas. The oven was programmed to rise from an initial temperature of 180–250°C at rates of

**Figure 4.** *Growth curves of Halamphora sp., D. salina and P. versicolor cultured batchwise.*

10°C min−1 (from 180 to 220), 2°C min−1 (from 220 to 240), and 5°C min−1 (from 240 to 250). Individual FAMEs were identified by comparing retention times with those obtained with laboratory standards and the manufacturer's instructions (Supelco).

### **3.7 Growth kinetics of three microalgae**

The four growth phases—lag, exponential, stationary and decline growth phases—are only observed on growth curves of *Halamphora* sp. (**Figure 4**). While *Halamphora* showed a short lag phase of 2 days, this phase was absent for *Phormidium* and *Dunaliella*. The exponential growth phase was observed for all the microalgae under continuous light but with different slopes. *Phormidium* and *Halamphora* grew faster with similar exponential phase about 5–6 days and reached maximum yield of 2.66 µg. ml-1 and 10.22 × 106 cells. ml-1, respectively at 8th day. During the exponential phase, the specific growth rate (µ) was about 2.40 and 2.15 day-1 for both *Phormidium* and *Halamphora*, respectively. However, it did not exceed 0.7 day-1 for *Dunaliella salina*. The growth rate of microalgae is very sensitive to culture conditions, such as irradiance and photoperiod limitation [45]. The density of *Halamphora* sp. is higher than those of *Halamphora acutiuscula* (5.91 × 105 cells. ml-1) and *Halamphora coffeaeformis* (6.17 × 105 cells. ml-1) which they are cultured in artificial sea water under light-dark (14/10h) cycles at a photon flux density of 300μmol photons m−2 s−1 [46].

For *Dunaliella salina*, the maximum cell density was recorded at 10th and did not exceed 1.65 × 106 cells. ml-1 (**Figure 4**). This value is higher than that reported in solar saltern by Elloumi et al. [47]. Guermazi et al. [31] stated that *D. salina* reached 6 × 106 cells. ml-1 when reard under 12h/12h light dark regime. Moreover, all microalgae curves are characterized by a short stationary phase (**Figure 4**). It seems that nutrients composition of the culture medium need to be optimized in order to maintain the cells at stationary phase.

#### **3.8 Physicochemical characterization of three microalgal species**

Microalgae could be easily grown in a laboratory and used for large-scale cultivation in bioreactors with the ability to control the quality of the cultures by providing purified culture medium that is free of toxic substances. Therefore, microalgae provide a more accessible way to produce qualitative biomolecules of interest [48–50]. Physicochemical characteristics of *D. salina*, *Halamphora* sp. and *P. versicolor* are presented in **Table 2**. The biomass of these microalgae contains moderate amounts of lipids, proteins, carbohydrates and an important percentage of chlorophyll a and carotenoids. The 7% dry matter content of *Halamphora* sp. is close to that found for other strains: 8% for *Halamphora* sp. [51], and for *P. versicolor* content 13% similarly with Singh, Parmar and Madamwar [52], who showed that the dry matter content of *Phormidium ceylanicum* is 10%*.* However, the lipids and proteins content of *Halamphora* sp. were relatively lower than the values published for other strains of *Halamphora* [53], and it was higher than that of *Amphora coffeaformis* [54]. For *D. salina*, the total lipids increased during growth hereas the amounts of proteins and sugars decreased, while for *P. versicolor*, it recorded a high level of protein (45%). The total sugars content of *Halamphora* sp. was 12.60% DW, which is consistent with that of some microalgae (5–23% DW) [55] and of *P. versicolor* was 21.56%. Moreover, these three algal species were found to be rich in chlorophyll, mainly chlorophyll a, and carotenoids. Continuous illumination favored also the synthesis of these pigments in *D. salina.* In fact, the synthesis of pigments was also stimulated under the

*The Solar Saltern of Sfax: Diversity of Hyperhalophilic Microalgae Species as a Promising... DOI: http://dx.doi.org/10.5772/intechopen.104712*


*Data are expressed as mean ± standard deviation of triplicates. FW: fresh weight; DW: dry weight; GAE: gallic acid equivalent; − not realized.*

#### **Table 2.**

*Physicochemical characteristics of three microalgae species.*

effect of light and allowing sufficient photosynthetic activity to be maintained for the synthesis of glycerol [56]. Our results also show that the 80% ethanolic extract of Bacillariophyceae and cyanobacterium the highest phenols and flavonoids contents. These high levels may be due to the culture conditions under the high salinity of 80 psu and the extraction conditions. Additionally, a high production of phycocyanin has been proven by the blue microalgae *P. versicolor* content 13%. These results are consisting with the observation by Singh, Parmar and Madamwar [52]. With respect to the ash content of *Halamphora* sp. (37.78% DW) (**Table 2**), it is in line with that found for another *Amphora* strain. Ash content exceeds 50% (55.8 to 67.9%) of the dry weight for some diatoms [54]. *Halamphora* sp. From Sfax solar salter has moderate amounts of sodium, potassium, calcium, and magnesium (**Table 3**). According to Boulay, Abasova, Six, Vass and Kirilovsky [57], the different species of microalgae do not develop the same strategies in order to survive under stressful conditions. We can assume that the species we studied might be a potential candidate for the production of biomolecules for pharmacological purposes.


#### **Table 3.** *Mineral content of Halamphora sp. [19].*


#### **Table 4.**

*Percentage of fatty acids composition of Halamphora sp. and D. salina reared in laboratory [19, 31].*

#### **3.9 Fatty acids composition of** *Halamphora* **sp. and** *D. salina*

The fatty acid profile of *Halamphora* sp. and *Dunaliella* sp. was composed of saturated (SFA), monounsaturated (MUFA), and polyunsaturated fatty acids (PUFA) which differed significantly from an alga to another (**Table 4**). The level of SFA recorded in *Halamphora* sp. and *Dunaliella* sp. is high, averaging 41.308 and 35.90% of total fatty acid, respectively. The pattern of SFA show that *Halamphora* is richer in SFA than *Dunaliella*. However, *Dunaliella* and *Halamphora* recorded a high level of palmitic acid (16:0) which accounted 21.0 and 27.42%, respectively. Hence, *Halamphora* sp. could be a suitable producer of SFA, which are easily convertible to biodiesel [58].

Moreover, *Halamphora* exhibited a high amount of palmitoleic acid (C16:1) which reached 45.089%, while that of *Dunaliella* did not exceed 2.2% of total FA. High levels of palmitoleic acid and other bioactive fatty acids were also detected in the fusi form morphotype of the Bacillariophyceae [59]. For *D. salina*, MUFAs were represented by 14:1 (n-5), 16:1 (n-7) and 18:1 (n-9). *D. salina* is an important source of 18:1 (n-9), whichreached14.9 ± 1.2%.

*The Solar Saltern of Sfax: Diversity of Hyperhalophilic Microalgae Species as a Promising... DOI: http://dx.doi.org/10.5772/intechopen.104712*

*Dunaliella* is rich in PUFAs with a percentage of 28.66%, while those of *Halamphora* did not exceed 6% of total fatty acid (**Table 4**). While *Halamphora* produced a noticeable level of EPA (2.367%), *Dunalila* is an important source of DHA reaching 4.3% of total fatty acid. Indeed, it is known that EPA is an important PUFA for health protection from many pathologies, including cardiovascular diseases [60] and cancer [61]. These PUFAs are known to have a number of important nutritional and pharmaceutical applications [62, 63]. They are also known to have beneficial effects on the health of human beings and to play a major role in the prevention of medical disorders in three areas: the heart and the circulation [64], inflammatory conditions and cancers, in particular colon tumorigenesis [65].
