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This report presented all the referring information about the organic culture of microalgae Dunaliella salina, Teodorescu, 1905, produced in organic effluents of Cuban Fishing Industry, and in a synthetic organic medium (MIP-1) and in control (Johnson medium). Here were studied the possibilities possessed by its algae as high-added value, in food industry and pharmaceutical products. A comparative study is offered on 69 bullfights (57 at laboratory scale and 8 in pilot plant), with different combinations and offered an economic valuation on the production of the algal bulk in form of paste in a pilot plant and to industrial scale as well as some possibilities for its use. This report suggest that the culture medium MIP-1 resulted advantageous, which is capable of achieving algae concentrations about 500,000 to 870,000 cell/mL during 15 days of be inoculated, represented a yield between 1,7 and 2,2 g/L on the dry weight base. According to the result data from the culture of this microalgae, to produce 1 kg of wet bulk was calculated a cost that fluctuates between $6.40 and 6.50, where the culture means in the case of the MIP-1 alone represents $0.09/m3 of culture medium.
Centro de Investigaciones Pesqueras, Ministerio de la Industria Alimentaria, Barlovento. Sta. Fe. Playa, Cuba
Teresita de Jesús Romero López
Centro de Investigaciones Hidráulicas (CIH), Universidad Tecnológica de La Habana, Cuba
*Address all correspondence to: gerardoeloy650@gmail.com
1. Introduction
This research is in a pilot’s plant phase and with the same one it is sought to interpret the behavior of the growth, carotenogenesis and protein contain of the microalga Dunaliella salina cultivated in synthetic medium with addition of a source of inorganic or organic carbon. The growth was measured with the purpose of evaluating its development under different culture conditions, since this can facilitate the decrease of the production costs in a climbed bigger. With the same principle the carotenogenosis was evaluated, to look for that fertilizer or which are those that increase the concentration of this pigment so important for the human health for its high nutritious value. If one wants to achieve a quick growth and development of this algae is an indispensable requirement the employment of near areas to salines, fundamentally those that are not subjected to the commercial exploitation of the salt, to have bigger area readiness, what represented a bigger net production. The culture medium that intends with this work is since of novel character, until the moment it has been planted in the existent bibliography that this microalgae species is not able to grow in a nutritive medium that its base of carbon is organic, reason because it is the first time that is cultivated in this way.
Among their perspectives uses, the culture in areas near natural saline, is the best option for commercial exploitation, also as high rate ponds. The lack of vitamins in the human feeding is a problem of world character, for what is indispensable to increase the sources of these, to achieve the man’s bigger survival. A way of achieving this end is with the culture of D. salina, which is able to accumulate a great quantity of Beta-carotene, a biological precursor of the vitamin “A”, which transforms it in this when are being ingested by the man and it is not toxic to the human organism like in the case of the ingest of synthetic vitamin “A” pills; for what that biological practice is of fundamental importance.
In the current world, the microalgae cultivation is of great interest, due to the uses but diverse that one makes of these, since they can be used in the production of food animal and human, as aide of high value, as chemical and biochemical products, as fertilizer and in the purification of polluted waters, among others [1, 2].
The microalgae D. salina that belong to the class Chlorophyceae and to the order Dunaliellales is of great importance for the man, since it is the fundamental natural source of the ß-carotene or provitamin “A”. The ß-carotene is recognized by its high one to be able to as antioxidant, what is the same thing an anti-carcinogenic of having proven effectiveness that can be used also as coloring [3], in the alimentary industry (mayonnaise’s, pastry, bakery, soups, juices, jells, etc.).
At the present time several countries are devoted to the commercial exploitation of the D. salina, among them the main ones are Australia and Israel, both although they use different technologies, they have been able to obtain a sustained production of this microalga.
Australia, the main producer of natural ß-carotene, not uses salt lakes dedicated to the exploitation of the salt, with very little energy expense but with use of big extensions of lands of which they prepare with easiness in this country. Israel uses high-speed lagoons with land saving but with more energy expense. Other countries have also attempted new cultivation technologies to increase the production of this microalga with the smallest possible cost.
In Cuba we have been carried out several studies to find the solutions, as culture medium, but economic and offer a high yield, for this in the Fisheries Industry Ministry (MIP) the work has been guided in two ways: one with a medium of where the source of carbon is organic and it comes from the residual waters of the fishing industry and another with a inorganic carbon that tries to substitute the conventional nutrients for its use in the salines. This in turn can use a source of inorganic or organic carbon, in dependence of the requirements of the cultivation.
To know the effect of different synthetic culture medium, on the growth and development of D. salina, were carried out 57 testes, according to the established experimental design as it is shown in theTable 1. As control the modified Johnson medium (MJ), was used [4], that is it usually used for the commercial culture of this microalgae. The employee inoculates belongs to the strain HFI-1; obtained in the MIP by sexual crossing among a strain from the Algal Culture Collection (ACC) of the “Centro Nacional de Investigaciones Científicas” (CENIC) in Cuba, whose origin is Chilean and another (MUR-8) from ACC at Algal Biotechnology Laboratory (ABL) of Murdoch University, Western Australia. The seeding, was carried out adding a seed of 300,000 cel/mL in erlenmeyers flask of 200 mL with 100 mL of salininzated medium with addition of sodium chloride (NaCl), until obtaining a concentration of 20%. The culture mother’s cells were in the phase of exponential growth and Betacarotene production.
The incident illumination received on all the experimental flasks, was continuous, during all day, with fluorescent light tubes of 40 Watts and the rotation of the flasks facilitated that it’s received a mean value of 9. 85 Klux. The luminous intensity was measured with a battery lux meter with three work ranges.
All the experimental series to laboratory scale got ready with distilled water, as diluent of the reagents that conform the synthetic medium. According to the applied reaction, was used commercial sugar cane like source of organic carbon; also for a specific culture molasses was used and for another, sodium bicarbonate (NaHCO3) to carry out a comparison among the different experiments, diverse culture medium were used with the unlike reagents that compose the Johnson medium in an individual way.
Of the obtained results, with the purpose of checking to a bigger scale the best results, was carried out an experiment to pilot plant scale with a lagoon that contained 1000 liters of culture, whose dilution water was fresh water with addition of commercial salt until reaching a concentration of 20% (Weight/Volume).
Cell counts were determined daily by a Neubauer cell hemocytometer with 1 mm2 of useful area. The pigments ß-carotene and chlorophyll a and b, was extracted with acetone and were determined according to the standard method of the ABL [5], in a cellular button after centrifuging during 8 minutes to 4500 r.p.m. A volume of 5 mL of blended cultivation with 5 mL of distilled water (H2O), for this way to diminish the salinity of the sample and to avoid that the cells that lack rigid cellular membrane break, being ignored this way the spill of the liquid, plasmatic to the water of the means. The calculations were carried out according to:
Total carotene = ABS452 * 3.86 (Vol. extract/Vol. it shows)
Chlorophyll to = 11.93 * ABS664 - 1.93 * ABS647
Chlorophyll b = 20.36 * ABS647 - 5.50 * ABS664
The primary data of the different series of growth in number and the ß-carotene concentrations did not resist a test of normality, and when relating the logarithm of the variances of each data series against the logarithms of the stockings of the cel/mL counts, a pending “b” of 2.47 was obtained since all the values were normalized according to expression log10 (x + 1). The slope for the regression of the ß-carotene concentrations was 2.33; because the same transformation was applied to normalize the data. Total proteins were determinate following the method of Lowry et al. [6]. The dry weight was analyzed by gravimeter. The validity of the results was analyzed applying an analysis of variance (ANOVA) of double classification with Duncan and Tuckey tests using a significance level of 95% and were solved by means of the packages of programs StatWin 8.0 and Excel 2016.
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.
Figure 1.
The growth curves of 57 experimental series, salinity of 20%.
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 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 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.
Figure 2.
Experimental groups where the high rates of growth were presented.
Figure 3.
Analysis of hierarchical classification that it offered the cluster more effective.
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 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.
Figure 4.
Two tests at pilot plant level in a high-speed lagoon, with paddles.
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.
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).
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).
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.
The evaluation of the economic feasibility of the process of obtaining of the microalgas D. salina was carried out on the base of the experience gathered until that moment with the one made for the Chlorella sp. [12, 13], and keeping in mind the pilot plant capacity installed at “Industrial de alimentos” (INDAL).
In this calculations was considerate:
a—a volume of cultivation of 3 m3.
b—a yield of 0.8 kg of dry alga for m3 of culture medium.
c—a productivity of humid alga that represents a biomass from 1.6 to 2.1 kg/day.
d—a retention time of 15–20 days for each harvesting, representing a crop cycle every two weeks.
e—an available time of 300 days troops, foreseeing 60 days of bottom of technological requirement and other causes.
The methodology for these calculations were carries out using the pattern of economic evaluation created by the ABL of the University of Murdoch, Western Australia, being calculated the cost of a kg of alga for the pilot plant installed at INDAL industry, as well as for an industrial plant. In the Table 2 a summarized information of the calculation about the unitary cost is presented. The cost obtained to produce 1 kg of alga, as it has been calculated with the pattern in question it was among $6.4 and $4.0 USD.
Inputs
Pilot plant
Industrial plant
Number of lagoons
2.0
2.0
Total Area (m2)
19.4
720.0
Total Volume (m3)
1.9
216.0
Outputs
Pilot plant
Industrial plant
Annual productivity (ton)
1.4
52.0
Total Power centrifugation (kW/d)
1.8
82.76
Losses for evaporation (m3/año)
11.7
1798.8
Hours for lagoon crop (h)
1.5
13.7
Centrifugal required
1.0
3.0
Volume harvested by day (m3/día)
3.7
205.20
Volume harvested by year (m3/año)
1170
65304.4
Biomass harvested by day (Kg/day)
1.2
68.2
Total biomass harvested (Kg/year)
389
21718.5
Cost for nutritious ($)
0
0.03
Construction
Pilot plant
Industrial plant
Land preparation ($/ha)
50.0
5000.0
Cultivation system ($/ha)
838.0
50000.0
Crop system ($/ha)
2102.0
459.8
Contingency ($/ha)
30.2
29.6
Total capital ($)
3050.0
621936.0
Relative cost
Pilot plant
Industrial plant
Work (%)
0.01
2.7
Energy (%)
5.0
13.7
Maintenance (%)
6.2
70.3
Over heads (%)
7.00
5.8
Capital (%)
0.03
7.34
Operation
Pilot plant
Industrial plant
Energy cost ($/kWh)
0.02
0.07
Maintenance (% of capital)
5.0
10.0
Average cost yearly
Pilot plant
Industrial plant
Algae cost Kg ($)
6.4
4.0
Table 2.
Summarized information of the calculation about the unitary.
The fundamental culture medium for the commercial cultivation of the D. salina was the modified Johnson (Borowitzka, [4]); the Ben-Amotz 1 [7]; the Ben-Amotz 2 [9] and those of the MIP with urea and the sodium that are presented in this work. The reagents to prepare the different culture that can be used by D. salina are presented in the Table 3 and their respective unitary costs in the Table 4.
Reagents
1
2
3
4
5
g/l
mM
mM
g/l
g/l
MgCl2.6H2O
1.5
—
—
—
—
MgSO4.7HH2O
0.5
5
5
—
—
KCl
0.2
—
—
—
—
CaCl2.H2O
0.2
0.2
0.3
—
—
KNO3
1.0
0.75
0.5
—
—
NaHCO3
0.043
50
50
—
—
KH2PO4
0.035
0.2
0.2
—
—
NaNO3
—
—
—
—
—
CuCl2
—
0.001
—
—
—
Traces
10 ml/l
—
—
—
—
Sol. FeCl2
10 ml/l
—
—
—
—
NaCl
200
200
200
200
200
Sugar
—
—
—
0.1
0.1
Urea
—
—
—
0.2
—
PO4-S
—
—
—
0.05
—
Table 3.
Reagents to prepare the different culture medium.
Reagents
$/ton
Urea
260.0
PO4-S
239.0
KNO3
667.5
NaNO3
456.0
MgCl2
4400.0
KH2PO4
700.0
MgSO4
599.8
KCl
26200.0
CaCl2
480.0
NaHCO3
302.2
NaCl
33.0
Sugar
225.0
Culture Medium
$/m3
NaCl $/m3
Johnson
5.93
5.94
Ben-Amotz “1”
1.93
5.94
Ben-Amotz “2”
1.91
5.94
MIP (urea)
0.09
5.94
MIP (sodium)
0.22
5.94
Table 4.
The most economical reagents.
According to the unitary costs that are presented in the Table 4, for the cultivation means used for the microalgae D. salina the most economical is since in fact the one that offered better results in the production of ß-carotene, without considering the addition of alone NaCl it represents $0.09 USD, for each m3 of cultivation. The cost of the salt (NaCl) that ascends $5.94 USD, for m3 of cultivation represent an initial expense, because when recycling 95% of the medium after the harvesting of microalgae, alone it would be necessary to add 5% of this cost ($0.30/m3) to the represented value, but it must de adding to all the culture medium, because for the comparison, all they use it.
The addition of a nitrate source like NaNO3 or Urea, it increased the speed of growth of this microalgae species with relationship to other cultures employees for D. salina.
With the substitution of the nutrients proposed in this work it is possible to diminish the cycle of life of this species since about 15 days, less than the 20; that it is when is commercially harvested.
The concentration of ß-carotene obtained by means of the medium of proposed culture is at the same level of the yields that were obtained in the world with this species.
The protein levels for this microalgae on the base of dry weight are reported between 50 and 88%, what allows their use like source of animal or human feeding.
Yields were obtained among 1.6 and 2.1 kg/m3 of culture with the medium that used urea, superphosphate or sodium nitrate, in substitution of the Johnson medium.
The additions of sugar as source of organic carbon accelerate the growth and carotenogenesis in the cultivation of this species.
D. salina can grow and to be developed heterotrophic, besides autotrophic like are recognized by all the authors.
According to the used evaluation pattern, the production of 1 kg of alga, is considered as $6.50 USD and the culture medium proposed for the commercial production of D. salina without considering the addition of alone NaCl represent $0.09 USD for each m3 of cultivation.
It is recommended to use a synthetic culture medium constituted by Urea, superphosphate and sugar cane, like Cuban MIP-1 medium for the commercial exploitation of microalga D. salina, as alternative for their cultivation to great scale, in areas near to natural salines that it does not explode commercially and it would allow their constant production for the ß-carotene extraction or use like biomass protein for the human feeding or animal.
Due to the carotene concentrations that can be obtained of this strain, their cultivation is recommended to great scale, for its later extraction and commercialization.
We intends to use sugar cane, like source of carbon to accelerate the growth and the carotenogenesis of this species and to diminish their cycle of life to less than 20 days, that which will benefit their commercial exploitation.
We thank to all the workers of the fishing industry INDAL, especially for the help that they have lent us for the realization of this work, and especially to Prof. M. Borowitzka for his important comments.
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Written By
Gerardo Suárez Álvarez and Teresita de Jesús Romero López
Submitted: 08 March 2022Reviewed: 04 April 2022Published: 02 November 2022
Open access peer-reviewed chapter
