**3. Results and discussion**

In all the experimental series to laboratory scale, the temperature of the cultivation stayed between 25 and 30°C. The rotation of the different experimental flasks favored in the luminous intensity of the same ones (80000–140,000 lux). The growth curves of 57 experimental series, with a salinity of 20%, are shown in the **Figure 1**; where clearly it is understood that there is a separation of the same ones in 2 groups, in those which the growths improve they evidenced in those that contemplated the addition in common of urea. When carrying out an analysis of hierarchical classification with the strategy of the complete binding with the Euclidean distances, must be proven, the separation in two groups, one that understands to the cases 37; 38; 39; 40; 50; 51 and 52; all with inclusion of urea and other big with three subdivisions that they offered significant statistical differences among them according to an analysis of variance of double classification. The F calculated for the 57 treatments was of 7.29 and the F of chart of 1.34 with 95% of probability. For the effect of the time of reaction the calculated F was of 15.10 against an F of chart of 1.53; for what thinks about that significant statistical differences exist among them.

Of these groups, 12 cases were chosen (MJ; 10; 12; 19; 20; 33; 39; 40; 48; 49; 50 and 51) that represent the groups where the rates of more growth were presented as one observes in the **Figure 2**. For the condition MJ, a curve average of 5 repetitions

**Figure 1.** *The growth curves of 57 experimental series, salinity of 20%.*

was used where an ANOVA offered a smaller Fc 1.08, at 2.26 of chart to indicate that they did not have statistical differences at a level of 95% among them. The conditions 10; 12 and 33 represent to the experiments where the fundamental nutriment was the potassium nitrate (KNO3) and they embrace the highest values (33); half and under (12 and 10). The conditions 19; 20; 39 and 40 represent the experiment that the fundamental nutriment have possessed sodium nitrate (NaNO3) and finally the 48; 4; 50 and 51, those the urea was the fundamental nutritional component. With these 12 groups to be carried out an analysis of hierarchical classification that it offered the cluster that is presented in the **Figure 3**, and 2 subgroups was obtained, one that represents the use of the urea and the other one subdivided in three, has in an end to the group that uses the Johnson medium and the NaNO3 to reason of 0.5 g/L, to which unites the condition 33 that is constituted by potassium nitrate (KNO3) and superphosphate (PO4-S). Among these, there are others represented by the other nutritional medium as it was presented in the **Figure 2**. A factor that apparently has favored the acceleration of the speed of growth in number, is the addition of sugar like source of organic carbon. The osmoregulation of this microalga species depends fundamentally on the glycerol production that accumulates inside the own cell and

#### **Figure 2.**

*Experimental groups where the high rates of growth were presented.*

it allows him to survive drastic changes of salinity and bigger salinity the ß-carotene production it is bigger that in turn allows him to support bigger intensity of luminous radiation, but Ben-Amotz and Avron [7], had pointed out that the polysaccharides facilitate the glycerol synthesis for that, apparently the addition of sugar facilitates the whole process of the cycles of life and ß-carotene production.

In any case, any company dedicated to the commercial exploitation of the *D. salina* in the world uses the addition of carbon in its organic form, because traditionally they have thought about according to the revision given by Borowitzka and Borowitzka [8] and Ben-Amotz and Avron [7, 9] that this species is unable to use this source of carbon, being emphasized that the same, alone can use it in its inorganic form, for what this form constitutes something new for this species; being significant in all the experiments carried out in the MIP, those series that used the Johnson medium always reached never bigger values to 500,000 cel/mL and with the addition of sugar were arrived until almost 2,000,000 cel/mL, what represents a great advantage for obtaining of biomass protein.

With the obtained results at the laboratory scale, were carried out two tests at pilot plant level in a high-speed lagoon, with paddles and with a 1000 L of culture medium (**Figure 4**) where used medium was fresh water salinized up to 20% with NaCl to which was added 0.5 g/L of NaNO3 and NaHCO3 to the concentration of the Johnson

**Figure 3.** *Analysis of hierarchical classification that it offered the cluster more effective.*

medium in a case (0.043 g/L) and 0.1 g/L of sugar. In the other case urea was used to reason of 0.2 g/L; 0.02 g/L of superphosphate and 0.1 g/L of sugar. These two variants were adopted because they represent to the highest results obtained regarding the growth in number of the cells of *D. salina*. In the lagoon with NaNO3 the speed of growth from a beginning was bigger, to reach values from 800,000 to 900,000 cel/mL among the day's 8 and 14; but in the lagoon with urea and superphosphate, although the speed of growth was not presented so quick the cellular concentrations reached the 900,000 cel/mL equally, to the 14 days to arrive until near values to 1,200,000 cel/mL, to the 20 days, for what anyone of these culture can be used for the commercial exploitation of this microalga; since generally the companies that market it harvest to the 20 days of initiate the cultivation and with near concentrations to the 500,000 cel/mL.

The illumination in the experiments outdoors, in the lagoons to plant pilot's scale, had fluctuated between 120,000 and 180,000 lux, with picks at 12 in the day; indicating that the received illumination was adapted for the growth of this microalga species, according to the data that report Ben-Amotz et al. [10] and Borowitzka and Borowitzka [4].

Regarding the pH, it is understood that the same one during the first hours of the day this in the surroundings of 8.15 to be increased up to 8.3 as it lapses the day and this is product of the consumption of CO2 for the algae during the hours of light [11]. These values are also among those understood among the good ones for the development of this species [8].

In the case of ß-carotene production, concentrations were reached of up to 11 μg ß-carotene/mL, of them those that understood addition of KNO3 arrived up to 6.5 μg ß-carotene/mL, those that had alone addition of NaNO3 arrived up to 3.5 μg ß-carotene/mL, those that used Johnson medium, 8 μg ß-carotene/mL was reached, but those that used urea arrived to concentrations of 11 μg ß-carotene/mL, those represent the best option for the cultivation to commercial scale.

**Figure 4.** *Two tests at pilot plant level in a high-speed lagoon, with paddles.*

For the 12 groups obtained by means of the analysis of hierarchical classification, regarding the growth in number, that offered significant statistical differences with 95% of probability according an ANOVA of double classification, for the different types of used medium (Fc = 2.52 > Ft = 1.99) and for the days of cultivation (Fc = 10.35 > Ft = 2.25); the evolution of the production of ß-carotene was analyzed (**Figure 5**), of which is understood that the biggest concentration (10–12 of μg Bc/mL) of this pigment, it happened in the cases 49; 50 and 51 all with use of the urea and the superphosphate like nutritious medium, with sugar as organic carbon. Results that they did not offer significant statistical differences with the cultivation carried out with the Johnson medium, reason because it is feasible to use the urea to commercial scale. For the rest of the cases the levels of ß-carotene did not surpass the 3 μg ß-carotene/mL, being among them those that used NaNO3 that so good results offered for the production of biomass.

**Figure 5.** *Evolution of the production of ß-carotene, with the better results.*

Analyzing the production of carotene for the results of the cultures employees for the lagoons to pilot scale (**Figure 6**), the biggest evolution was reported for the lagoon with addition of urea (0.2 g/L); superphosphate (0.05 g/L) and sugar (0.1 g/L).

The content of proteins in this microalga strain fluctuated between 50 and 88%, being obtained the highest values with those that were in cultivation that it was added NaNO3. Concentration is it presents it like an alternative in the animal or human feeding.

The dry weight of the *D. salina* corresponded to values between 10 and 40 pg./cel, like to yields between 1.7 and 2.2 g of cells per liter of culture, which are inside the highest among those reported in the literature like commercial scale. The highest coincide with the culture those was added NaNO3 or urea and sugar.

*Mixotrophyc Culture of* Dunaliella salina *in Cuban Fishing Wastewaters DOI: http://dx.doi.org/10.5772/intechopen.104803*

#### **Figure 6.**

*Carotene for the lagoons to pilot scale, the biggest evolution was reported for the lagoon with addition of urea (0.2 g/l); superphosphate (0.05 g/l) and sugar (0.1 g/l).*
