Continuous Microalgal Cultivation for Antioxidants Production

Microalgae, including cyanobacteria, represent a valuable source of natural compounds that have remarkable bioactive properties. Each microalga species produces a mixture of antioxidants with different amounts of each compound. Three aspects are important in the production of bioactive compounds: the microalga species, the medium composition including light supplied and the photobioreactor design, and operation characteristics. In this study, the antioxidant content and productivity performance of four microalgae were assessed in batch and continuous cultures. Biomass productivity by the four microalgae was substantially enhanced under continuous cultivation by 5.9 to 6.3 times in comparison with batch cultures. The energetic yield, under the experimental conditions studied, ranged from 0.03 to 0.041 g biomass kJ−1. Phenols, terpenoids, and alkaloids were produced by Spirulina platensis, Isochrysis galbana, and Tetraselmis suecica, whereas tocopherols and carotenoids were produced by the four microalgae, except for phycocyanin and allophycocyanin, which were only produced by S. platensis and Porphyridium cruentum. The findings demonstrate that the continuous cultivation of microalgae in photobioreactors is a convenient method of efficiently producing antioxidants.


Introduction
Microalgae are a promising group of microorganisms that produce novel compounds, known as secondary metabolites. Value-added microalgal products are associated with the health and cosmetics industries, unrestricted products produced for medical care, and the aquaculture industry [1,2]. Cure from cancer is the goal of cancer therapy. Many deaths in women are due to cervical, stomach, and breast cancers; the demand for anticancer agents from natural sources has increased for antitumor therapy due to the less severe or lack of secondary effects of these treatments [3]. Various microalga species have been reported to produce nutraceuticals, renewable-energy-related compounds, β-carotene, astaxanthin, polyunsaturated fatty acids (PUFAs), and biocolorants; all these compounds are accumulated during growth and are produced for different applications such as health-and food-based products [1], wastewater treatment, aquaculture, etc. [2,4].
reported an engineering strategy to simultaneously produce PC with a productivity of 0.13 g L −1 d −1 and a CO 2 consumption of 1.58 g L −1 d −1 with Spirulina platensis [20].
Two kinds of photobioreactors exist that produce microalgae biomass: open and closed photobioreactor designs. The dynamics of the cultures allow them to be produced either in batches or continuously, working under natural or artificial light irradiance. However, open systems have two disadvantages: contamination with bacteria and light supply. Additionally, the general cell density is low and only few microalgae species are suitable for this production mode due to contamination. Closed systems are an alternative to producing highly valuable compounds such as antioxidants. The control of all operational parameters is more easily accomplished in closed systems, but costs are high. One alternative to enhancing biomass density and production is operating photobioreactors during continuous production. One difficulty to be overcome is the low growth rate of microalgae that may cause complications in achieving continuous cultivation before washout of the cells [17]. In the present study, antioxidants production by microalgae was assessed in continuous mode operation in a photobioreactor (PBR).

Growth Analyses in Batch and Continuous Cultivation
The growth profiles were analyzed in the batch and continuous cultures of four microalgae. The growth performance of four microalgae in batch cultivation is shown in Figure 1. Specific growth rates were calculated by the Gompertz growth equation, which were found to be 0.53, 0.49, 0.51, and 1.1 d −1 for Spirulina platensis, Isochrysis galbana, Tetraselmis suecica, and Porphyridium cruentum, respectively. Biomass concentration and biomass productivity values are summarized in Table 1. The biomass concentrations in batch cultures were 3, 1.5, 1.8, 2.1 g L −1 for S. platensis, I. galbana, T. suecica, and P. cruentum, respectively ( Table 1). The growth of microalgae depends on the culture conditions such as medium composition, hydrodynamics in the bioreactor, and light irradiation that reaches every cell. By comparing all data of the Gompertz growth parameters among all microalgae, µ (growth specific rate) and maximum biomass density were different for all strains (Table 1).
Molecules 2020, 25, x FOR PEER REVIEW 3 of 17 Two kinds of photobioreactors exist that produce microalgae biomass: open and closed photobioreactor designs. The dynamics of the cultures allow them to be produced either in batches or continuously, working under natural or artificial light irradiance. However, open systems have two disadvantages: contamination with bacteria and light supply. Additionally, the general cell density is low and only few microalgae species are suitable for this production mode due to contamination. Closed systems are an alternative to producing highly valuable compounds such as antioxidants. The control of all operational parameters is more easily accomplished in closed systems, but costs are high. One alternative to enhancing biomass density and production is operating photobioreactors during continuous production. One difficulty to be overcome is the low growth rate of microalgae that may cause complications in achieving continuous cultivation before washout of the cells [17]. In the present study, antioxidants production by microalgae was assessed in continuous mode operation in a photobioreactor (PBR).

Growth Analyses in Batch and Continuous Cultivation
The growth profiles were analyzed in the batch and continuous cultures of four microalgae. The growth performance of four microalgae in batch cultivation is shown in Figure 1. Specific growth rates were calculated by the Gompertz growth equation, which were found to be 0.53, 0.49, 0.51, and 1.1 d −1 for Spirulina platensis, Isochrysis galbana, Tetraselmis suecica, and Porphyridium cruentum, respectively. Biomass concentration and biomass productivity values are summarized in Table 1. The biomass concentrations in batch cultures were 3, 1.5, 1.8, 2.1 g L −1 for S. platensis, I. galbana, T. suecica, and P. cruentum, respectively ( Table 1). The growth of microalgae depends on the culture conditions such as medium composition, hydrodynamics in the bioreactor, and light irradiation that reaches every cell. By comparing all data of the Gompertz growth parameters among all microalgae, μ (growth specific rate) and maximum biomass density were different for all strains (Table 1).     Biomass productivity (XVD (g biomass L −1 d −1 ), where X is biomass (g), V is working volume (L −1 ), and D is dilution rate (d −1 )) obtained in terms of total light supplied (I 0 A (kJ d −1 ) energy supplied from light to a surface area per unit of time) is shown in Figure 2. The productivity of biomass at a light intensity of 39.63 kJ d −1 in batch culture was 0.34, 0.24, 0.2, and 0.16 g biomass L −1 d −1 (Table 1) and 2.01, 1.58, 1.16, and 0.95, g biomass L −1 d −1 , for S. platensis, P. cruentum, T. suecica, and I. galbana, respectively, in continuous cultivation ( Figure 2). Comparison of the biomass productivity (XVD) though linear regression analysis of each microalga showed statistically significant differences with increasing light intensity. The productivity increase of biomass concentration was 5.9-6.6 times higher in continuous cultures, which may support the final quantity of antioxidants produced, as shown below. The energetic yield of the produced biomass for the supplied light energy varied according to the strain: 0.041, 0.037, 0.033, and 0.030 g biomass produced per kJ for S. platensis, P. cruentum, T. suecica, and I. galbana, respectively, and a yield of conversion efficiency (Y ce , luminous energy to chemical energy) of 1.04, 0.95, 0.83, and 0.75, respectively (-∆Ho × Y kcal ; enthalpy change for O 2 = 106 kcal mol −1 O 2 ). Molecules 2020, 25, x FOR PEER REVIEW 5 of 17

Antioxidant Production in Batch Culture
Total phenols, alkaloids, and terpenoids content were measured in S. platensis, I. galbana, and T. suecica during batch cultivation ( Table 3). All compounds were produced by the microalgae in significantly different amounts (p < 0.05), except for alkaloids between I. galbana and T. suecica, where no significant differences were observed. Phenol, alkaloids, and terpenoids were more abundant in S. platensis than the others; these compounds accumulated during growth. The productivities of phenols, alkaloids, and terpenoids showed significant differences among the three species ( Figure 3). The productivity of phenols in S. platensis was four times that of I. galbana and 1.75 times that of T. suecica (Figure 3). Alkaloids and terpenoids also showed significant differences among the three species of microalgae.

Antioxidant Production in Batch Culture
Total phenols, alkaloids, and terpenoids content were measured in S. platensis, I. galbana, and T. suecica during batch cultivation ( Table 3). All compounds were produced by the microalgae in significantly different amounts (p < 0.05), except for alkaloids between I. galbana and T. suecica, where no significant differences were observed. Phenol, alkaloids, and terpenoids were more abundant in S. platensis than the others; these compounds accumulated during growth. The productivities of phenols, alkaloids, and terpenoids showed significant differences among the three species ( Figure 3). The productivity of phenols in S. platensis was four times that of I. galbana and 1.75 times that of T. suecica ( Figure 3). Alkaloids and terpenoids also showed significant differences among the three species of microalgae. Tocopherols (α, δ, and γ), carotenoids, superoxide dismutase (SOD), and phycobiliproteins were recorded during batch production in the four microalgae ( Table 4). The total tocopherol amount is reported here as the sum of α, δ, and γ tocopherols in I. galbana, T. suecica, as well as the sum of α and γ tocopherols in S. platensis and P. cruentum. In S. platensis and P. cruentum, δ-tocopherol was not found. A large amount of tocopherol was produced in P. cruentum, with αand γ-tocopherol at 80.5 and 104.2 µg g −1 , respectively. Durmaz et al. reported 106.3 µg g −1 biomass for both αand γ-tocopherols in P. cruentum [21], which is different from the 185 µg g −1 obtained in this study. Figure 4 shows the accumulation of PC and APC in S. platensis during growth. Molecules 2020, 25, x FOR PEER REVIEW 6 of 17 Tocopherols (α, δ, and γ), carotenoids, superoxide dismutase (SOD), and phycobiliproteins were recorded during batch production in the four microalgae ( Table 4). The total tocopherol amount is reported here as the sum of α, δ, and γ tocopherols in I. galbana, T. suecica, as well as the sum of α and γ tocopherols in S. platensis and P. cruentum. In S. platensis and P. cruentum, δ-tocopherol was not found. A large amount of tocopherol was produced in P. cruentum, with α-and γ-tocopherol at 80.5 and 104.2 μg g −1 , respectively. Durmaz et al. reported 106.3 μg g −1 biomass for both α-and γtocopherols in P. cruentum [21], which is different from the 185 μg g −1 obtained in this study. Figure  4 shows the accumulation of PC and APC in S. platensis during growth.    The productivities of antioxidants in microalgae are shown in Table 5. The productivity of tocopherols in S. platensis and P. cruentum showed no significant differences. Carotenoids, PC, APC, and SOD productivities were statistically different in all microalgae.

Strain
Tocopherols  The productivities of antioxidants in microalgae are shown in Table 5. The productivity of tocopherols in S. platensis and P. cruentum showed no significant differences. Carotenoids, PC, APC, and SOD productivities were statistically different in all microalgae. The same letter in each column indicates no significant difference in the means of 3 replicates (p < 0.05). nd: not determined.

Antioxidant Concentration and Productivities in Continuous Culture at Different Light Intensities
The concentrations of total phenols, terpenoids, and alkaloids were analyzed in continuous cultures of S. platensis, I. galbana, and T. suecica at different light intensities. Total phenols, alkaloids, and terpenoids concentrations were significantly different among the three microalgae (p < 0.05; Table 6). Phenols, alkaloids, and terpenoids content did not significantly change in any of the microalgae with 16.27 and 21.26 kJ d −1 of supplied light intensity (Table 6). By increasing light intensity to 30.42 kJ d −1 phenols, alkaloids, and terpenoids content changed significantly in every microalga, except for phenols content in I. galbana. Phenols, alkaloids, and terpenoids content showed significant differences at 39.63 kJ d −1 in comparison with the contents of these compounds at 16.27 and 21.26 kJ d −1 in every microalga. A significant difference in phenols content was found in S. platensis and I. galbana by changing light intensity from 30.42 to 39.63 kJ d −1 (Table 6). The productivity of phenols, alkaloids, and terpenoids in S. platensis, I. galbana, and T. suecica under four different light intensities cultured under continuous cultivation is shown in Figure 5A-C. The three compounds' productivity increased significantly with increasing light intensity in every microalga. Phenol productivity in S. platensis was five times higher than in I. galbana, and more than twice that of T. suecica at a light intensity of 39.63 kJ d −1 . Phenol productivity increased more than twice in S. platensis, more than four times in I. galbana, and three times than T. suecica with the change in light intensity from 16.27 to 39.63 kJ d −1 (Figure 5A). Alkaloid productivity of S. platensis was more than three times that of I. galbana, and more than four times that of T. suecica ( Figure 5B). The alkaloid productivity was 2.1 times higher in S. platensis, 4.1 times in I. galbana, and more than three times higher in T. suecica with increasing the light intensity from 16.27 to 37.63 kJ d −1 (Figure 5B). Terpenoids productivity increased significantly with increasing light intensity. The productivity of terpenoids in S. platensis was significantly different from the productivity in I. galbana and T. suecica, whereas productivity of terpenoids in I. galbana and T. suecica showed no significant differences ( Figure 5C).  Tocopherols content was significantly different in the four microalgae analyzed. T. suecica and P. cruentum showed higher amounts of tocopherols than S. platensis and I. galbana. In addition, tocopherols concentration increased with light intensity in the four microalgae (  Tocopherols productivity in S. platensis and P. cruentum was not significantly different at 16.27 and 21.26 kJ d −1 of light intensity supplied, but significant different when light intensity increased from 21.26 to 39.63 kJ d −1 . P. cruentum showed a productivity increase of more than 1.2 times the productivity of S. platensis, 2.66 times the productivity of I. galbana, and 1.57 times the productivity of T. suecica ( Figure 6). Tocopherols content was significantly different in the four microalgae analyzed. T. suecica and P. cruentum showed higher amounts of tocopherols than S. platensis and I. galbana. In addition, tocopherols concentration increased with light intensity in the four microalgae (  Tocopherols productivity in S. platensis and P. cruentum was not significantly different at 16.27 and 21.26 kJ d −1 of light intensity supplied, but significant different when light intensity increased from 21.26 to 39.63 kJ d −1 . P. cruentum showed a productivity increase of more than 1.2 times the productivity of S. platensis, 2.66 times the productivity of I. galbana, and 1.57 times the productivity of T. suecica ( Figure 6).  The carotenoids content in the microalgae changed significantly from light intensities of 16.27 to 39.63 kJ d −1 . Carotenoid content increased 1.5, 1.35, 1.56, and 1.57 times in S. platensis, I. galbana, T. suecica, and P. cruentum, respectively. At a light intensity of 39.63 kJ d −1 , carotenoids content showed no significant differences among the microalgae studied (Table 8). Carotenoids productivity changed with the biomass productivity for each microalga; S. platensis had a productivity at least twice that of I. galbana and T. suecica and at least 1.3 times that of P. cruentum The amounts of UA SOD were significantly different among S. platensis, I. galbana, and P. cruentum at different light intensities, but no difference was observed between S. platensis and T. suecica. SOD units increased significantly by increasing light intensity supplied in each microalga (Table 9). Identical letters indicate no statistic differences in the means of 3 replicates (p < 0.05).
The SOD UA productivity increased in accordance with biomass productivity with increasing supplied light energy (Figure 8). Soluble protein was found to be 65.7, 43.5, 51.3, and 77.9 mg protein The amounts of UA SOD were significantly different among S. platensis, I. galbana, and P. cruentum at different light intensities, but no difference was observed between S. platensis and T. suecica. SOD units increased significantly by increasing light intensity supplied in each microalga ( Table 9).
The SOD UA productivity increased in accordance with biomass productivity with increasing supplied light energy (Figure 8). Soluble protein was found to be 65.7, 43.5, 51.3, and 77.9 mg protein g −1 biomass for S. platensis, I. galbana, T. suecica, and P. cruentum, respectively. SOD UA productivity was significantly different in all microalgae.   The PC production by S. platensis was 7.0 times that by P. cruentum; APC production of S. platensis was 8.9 times that of P. cruemtum (Figure 9). Although PC and APC content decreased with increasing light intensity, the production of both pigments increased with increasing light intensity due to biomass productivity.  Phycocyanin (PC) and allophycocyanin (APC) were found only in S. platensis and P. cruentum. Compared with the other antioxidants analyzed in this work, PC and APC content decreased with increasing light intensity (Table 10). PC concentration in S. platensis was 5.6 times the concentration in P. cruentum, and APC concentration in S. platensis was 7.1 times the concentration of APC in P. cruentum at a light intensity of 39.63 kJ d −1 . The PC production by S. platensis was 7.0 times that by P. cruentum; APC production of S. platensis was 8.9 times that of P. cruemtum (Figure 9). Although PC and APC content decreased with increasing light intensity, the production of both pigments increased with increasing light intensity due to biomass productivity.
The PC production by S. platensis was 7.0 times that by P. cruentum; APC production of S. platensis was 8.9 times that of P. cruemtum (Figure 9). Although PC and APC content decreased with increasing light intensity, the production of both pigments increased with increasing light intensity due to biomass productivity. The extract of S. platensis was dissolved in solvent, then the extract was analyzed by FTIR (Table  11). Several signals appeared related to some characteristic vibrational groups. An important vibrational signal, the methylene group (2829-2848 cm −1 [22]), appeared at 2835 cm −1 , which is possibly found in carotenoids and phytosterols molecules such as lycopene, which is a precursor of The extract of S. platensis was dissolved in solvent, then the extract was analyzed by FTIR (Table 11). Several signals appeared related to some characteristic vibrational groups. An important vibrational signal, the methylene group (2829-2848 cm −1 [22]), appeared at 2835 cm −1 , which is possibly found in carotenoids and phytosterols molecules such as lycopene, which is a precursor of carotene [23]. Another vibrational signal found at 1230 cm −1 was assigned to the phenyl group and 1517 cm −1 to the stretching of the flavone phenyl ring; these signals correlate well with antioxidants such as flavone. The vibration band at 1740 cm −1 corresponds to the carbonyl group of phytosterols and the signal at 1612-1652 cm −1 to the C=O group, which are signals assigned to ketones found in flavonoids. Table 11. FTIR analyses of S. platensis extracts. Signal vibrations found in the S. platensis extract dissolved in solvents.

Discussion
The microalgae studied in this work exhibit reliance on antioxidant production on the growth conditions; however, strain species is an important factor. Antioxidant accumulation occurs along with cell growth, playing important roles in microalgal cells.
Phycocyanin has different biotechnological applications in cosmetics, food, and feed industries, analytical biochemistry, and therapeutic applications [19]. Zheng et al. [19] reported PC-protection against nephropathy by inhibiting oxidative stress, perhaps due to the similarity of the PC chemical structure with that of biliverdin. Chen et al. [20] reported a strategy to simultaneously improve biomass and PC productivities in a flat PBR with batch cultivation of 0.74 and 0.125 g L −l d −1 under a light intensity of 700 µmol m −2 s −1 (32.95 × 10 −3 kJ cm −2 h −1 ) [20], which is close to double the 15.1 × 10 −3 kJ cm −2 h −1 (320 µmol m −2 s −1 ) light intensity used in the present work. Here, the biomass and PC productivities were 2 and 0.4 g L −1 d −1 , respectively, which are 2.7 times the biomass and 3.2 times the PC productivity at 320 mmol m −2 s −1 compared to the values reported by Chen et al. [20] at 700 µmol m −2 s −1 . Although PC content in S. platensis was 235 PC mg g −1 biomass at 132 µmol photons m −2 s −1 and 198 PC mg g −1 biomass produced at 320 µmol m −2 s −1 , PC productivity at 320 µmol photons m −2 s −1 was 1.7 times the PC produced at 132 µmol m −2 s −1 . Zheng et al. [19], in a PBR with a light intensity of 200 µmol m −2 s −1 in batch culture, obtained 0.39 g L −1 d −1 biomass productivity for S. platensis [19]. Schipper et al. worked with Leptolyngbya sp. to produce PC in continuous culture at 80 µmol photons m −2 s −1 . Under these conditions, 86 mg of PC g −1 biomass was produced with a productivity of 78.8 mg L −1 d −1 [24].
Microalgae produce important antioxidants: alkaloids, terpenoids, phenols, tocopherols, carotenoids, phycobilins, and other pigments and enzymes, which are involved in active antioxidant treatments due to their anti-inflammatory, antibacterial, and anticancer, lipid peroxidation activities. Porphyridium cruentum produced another promising compound, sulfate-polysaccharide, which has either anti-inflammatory or antiallergic properties. α-tocopherol is the most abundant with the highest antioxidant activity in terrestrial plants; it functions in the electron transport reactions and cell membrane stabilization functions [25]. Animals are unable to synthesize tocopherols, so plant sources are a primary choice for human consumption. Nevertheless, microalgae have become a natural and alternative source of tocopherols, containing other tocopherols that provide remarkable synergistic antioxidant activity [18]. Durmaz et al. [21] reported 55.2 and 51.3 µg g −1 biomass of αand γ-tocopherol, respectively, in P. cruentum [21]. The amounts of αand γ-tocopherols (80 and 104 µg g −1 biomass, respectively) in P. cruentum found in this study are in agreement with those reported by Durmaz et al. [21]. In addition, δ-tocopherol was found in T. suecica, which produces γ-tocopherol in considerable amounts, but also produced α-tocopherols. Spirulina maxima produces αand γ-tocopherols [18], but the amounts are lower than in S. platensis in this study.
Carotenoids and their derivative compounds bond to lipoproteins and play an important role as antioxidants during photosynthesis; they have been associated with potential protection against lipid peroxidation, DNA and protein oxidation, and in cancer prevention [8]. Photosynthetic organisms contain carotenoids; particularly in microalgae, α-, and β-carotenes are present, whereas other microalgae are important axthasantin producers. The carotenoid content in microalgae rarely surpasses 10% and they are present as a mixture of carotenoids. In P. cruentum, Rebollo et al. [26] found 1.0 mg g −1 biomass, whereas Santiago et al. reported 4.6 mg g −1 biomass for β-carotene in T. suecica [18].
SOD has been found in S. maxima, Nannochloropsis sp., T. suecica, Chaetoceros sp., Synechoccocus, and P. cruentum, with different amounts in each microalga. In T. suecica and P. cruentum, activities of 20.4 and 58 UA mg −1 protein were measured, respectively [18], in comparison with 54.6 and 74.5 UA mg −1 protein reported in this work, respectively. The culture conditions may affect the formation of antioxidant compounds, in particular SOD; this enzyme may be induced in response to the increase or demand to drive reactive oxygen species generated by an increase in photosynthesis activity.
The FTIR spectra of S. platensis extracts showed vibrational signals related to some antioxidant molecules, such the signal at 2835 cm −1 assigned to the methylene group, at 1230 cm −1 to the phenyl group, the stretching at 1517 cm −1 assigned to the flavone phenyl ring, and the vibrational signals at 1612-1652 cm −1 assigned to the C=O ketone groups found in flavonoids.
Microalgae extracts containing a mixture of antioxidants have the capacity to scavenge superoxide anions, hydrogen peroxides, and hydroxyl radicals [18]. The antioxidant machinery involves enzymatic and nonenzymatic antioxidants. These extracts also prevent oxidative damage by efficiently neutralizing free radicals, which includes catalase by the conversion of hydrogen peroxide into water and molecular oxygen. Other enzymes do not neutralize free radicals directly but create a reducing environment. Nonenzymatic antioxidants include vitamin E, cofactor Q 10 , peptides (glutathione), phenols, and pigments [7]. All these antioxidants are synthesized in microalgae in different combinations and concentrations, highly dependent on strain, culture medium composition, time of harvesting, bioreactor geometry, and irradiated surface. The contents of antioxidants found in microalgae provide the possibility to focus efforts on obtaining reliable sources of antioxidants for production in biofactories.

Microalgae Strains, Culture Medium and Growth Conditions
The microalgae Porphyridium cruentum, Isochrysis galbana, Tetraselmis suecica, and Spirulina platensis were acquired from our own culture collection of microalgae. For I. galbana, T. suecica, and P. cruentum, medium "f" [27] of Guillard and Ryther was used. S. platensis was grown on SOT standard medium [28]. Cultivation was performed in a jar-fermenter (Applikon Biotechnology BV, JG Delft, The Netherlands) equipped with 2 Rushton impellers 60 mm in diameter and a working volume of 1.0 L. The top surface of the reactor (illuminated surface) had a surface area of 109.36 cm 2 and diameter of 11.8 cm. Light (0.149, 0.194, 0.278, and 0.36 Jcm −2 d −1 , with an irradiated surface of 109.36 cm 2 , producing I 0 A = 16. 27, 21.26, 30.42, and 39.63 kJ d −1 , respectively) was supplied on the top of the bioreactor. The reactor was operated at a 30 ± 2 • C, agitation 200 rpm, and with an initial pH of 7.5 for Porphyridium cruentum, Isochrysis galbana, and Tetraselmis suecica, and pH 8.5 for Spirulina platensis. The illumination was supplied with white light LED lamps on the top of the reactor. The photobioreactor was covered with aluminum foil, assuming that light filled the entire volume of the culture. The bioreactor was connected to a reservoir containing fresh medium; the flow rate was controlled by a valve. The fresh medium was fed into the bioreactor and the broth exit valve was open at the same flow rate. Dilution rate (D) was achieved after at least three days without changes in the cell density in the bioreactor, then the culture was considered in continuous mode or in a steady state. Cells were harvested by centrifugation at 12,000× g for 10 min at 4 • C, then the collected biomass was kept at -20 • C if not used immediately. (Note: Conversions used: 1 lux = 1.55 µmol m −2 s −1 ; 1 cd = 10.76 lux; 1 cd = 7.85 × 10 −6 kJ cm −2 h −1 ), (I 0 A is defined as the energy from light supplied to a surface area per unit of time).

Quantitation of Phenols, Alkaloids, and Terpenoids
S. platensis, I. galbana, and T. suecica biomass (0.1-0.5 g) were suspended in 5.5 mL of 0.01 M phosphate buffer, pH 6.5, and 0.15M NaCl. The suspension was frozen at −20 • C, thawed and sonicated, and then left at 5 • C overnight. The suspension was centrifuged at 12,000× g for 15 min at 4 • C; the supernatant was considered crude extract. Total alkaloids was measured as follows: The crude extract was mixed with chloroform (1:1 v/v) and the chloroform phase was separated and concentrated. The concentrate was resuspended with 1 mL of chloroform, and 6.26 mL of H 2 SO 4 (0.02N) was added; chloroform was eliminated. Some drops of methyl red indicator were added into the solution, then the mixture was titrated with NaOH (0.02 N). Each mL of NaOH (0.02 N) added was equivalent to 5.78 mg of alkaloids. Total phenols was measured as follows: 0.1 mL of the extract was mixed with 2.8 mL of deionized water, 2 mL of sodium carbonate (2%), and 0.1 mL of Folin-Ciocalteu reagent (50%). The mixture was incubated for 30 min at room temperature and the absorbance of the mixture was recorded at 750 nm in a spectrophotometer [29]. Ferulic acid was used as the standard. For the determination of total terpenoids, 5 mL of extract was placed in an oven at 100 • C for 1 h. After cooling, 5 mL of freshly prepared vanillin reagent (0.7% in 65% H 2 SO 4 ) was added. The tubes were heated at 60 • C in a water bath for 1 h. After cooling on ice, the absorbance was measured at 473 nm. Saponin was used as the standard. For FTIR analysis, an FTIR spectrometer (Frontier model, PerkinElmer, Waltham, MA, USA) was used; the sample was redissolved with a mixture of acetonitrile/methanol (70:30).

Tocopherols and Carotenoids Determination
Biomass (0.1-0.15 g) was mixed with a 2 mL solution containing 60% ethanol, pyrogallol (6%), and 0.87 mL KOH (60%) under N 2 atmosphere, then it was sonicated. The mixture was incubated for 1 h at 70 • C in the dark, then the mixture was chilled on ice and 3.13 mL (NaCl 2%) was added and then extracted with n-hexane (1:1 v/v). The n-hexane was eliminated under N 2 . The concentrated extract was suspended in 0.7-1.0 mL of methanol and filtered through a 0.45 µm membrane, the extract was filtered and stored at −70 • C. The methanolic extract was used to measure tocopherols in HPLC [18]. Twenty microliters of the extract were passed through a Zorbax XDB-C8 (4.6 mm × 150 mm, 5 µm; Agilent Technologies, Santa Clara, CA, USA) column and eluted with an isocratic solution of methanol/water (95:05) at a flow rate of 1.0 mL min −1 . A fluorescence detector using an excitation λ 292 , an emission wavelength of λ 340 , and a UV detector at λ 340 were used to measure the tocopherols content [18]. Commercial α, δ, and γ-tocopherols served as the standards for quantification and detection. Total carotenoids were measured at 480 nm (A 1% = 2500 absorption coefficient) of absorbance in a UV-Vis spectrophotometer [30].

Superoxide Dismutase Activity Determination
We mixed 0.05 to 0.1 g of biomass with 1 mL phosphate buffer (0.2 M, pH 8.0, 5 mM EDTA, 1 mM DTT), sonicated for 2 min, then centrifuged at 12,000× g at 4 • C for 30 min. The supernatant was used for measurements or stored frozen. For SOD activity determination, the Bioxytech SOD-525 assay kit (Oxis International, Foster, CA, USA) was used. The reaction was followed at 525 nm at 30 • C for 1 min. The reaction contained 50 µg protein mL −1 of extract. Commercial SOD was used as the standard. Protein content was measured using Bio-Rad reagent (Bio-Rad Laboratories, Inc., Berkeley, CA, USA) at 595 nm in a spectrophotometer [18].

PC and APC Determinations
S. platensis and P. cruentum biomass (0.1-0.5 g) was suspended in 5.5 mL of potassium phosphate buffer 0.01 M, pH 6.5, and 0.15M NaCl. The suspension was frozen at −20 • C, thawed, and sonicated, then left at 5 • C overnight. The suspension was centrifuged at 12,000× g for 15 min at 4 • C. The supernatant contained phycocyanin-allophycocyanin extract. Phycocyanin and allophycocyanin concentrations were calculated according to Bennet and Bogard [31] by measuring the absorbance at 615 and 652 nm in a spectrophotometer and applying the following equations (data in mg mL

Statistical Analyses
GraphPad Prisma software version 8.4.3 (GraphPad Software, San Diego, CA, USA) was used for all analyses. For growth, the Gompertz model was used to calculate growth parameters. For biomass productivity at different light intensities, a linear regression analysis was applied and we compared the 4 strains. To compare the concentration and productivity of all strains at different light intensities, Tukey's multiple comparison test was applied (p < 0.05).

Conclusions
Microalgae, including cyanobacteria, are manipulatable organisms that grow in photobioreactors, where the medium composition, light intensity, reactor design, reactor operation, metabolism, and microalga species are important factors affecting the production of compounds synthesized by the microalgae. In this work, well-known microalgae species that produce secondary metabolites were assayed for the production of different compounds that have remarkable antioxidant activity. We found that the microalgae produce a mixture of antioxidants in different amounts depending on the species, light supplied, and bioreactor operation. Phenols, alkaloids, terpenoids, tocopherols, carotenoid, PC, and APC content, including an enzyme, SOD, were produced by the microalgae tested. Continuous bioreactor operation substantially improved biomass and antioxidant productivity in comparison with batch cultivation in the same bioreactor under the same conditions, except that continuous cultivation produces the same amount of biomass and secondary metabolites every day over nine days of cultivation as achieved in batch cultures. Continuous cultivation drives the microalgal cell metabolism of biomass and metabolites synthesis in the bioreactor; the mathematical description of continuous cultivation is referred to as a steady state, where biochemical pathways constantly dynamically synthesize molecules. Antioxidants are produced naturally as a mixture. We deduced that the combination of continuous culture with the correct nutrient limitation will improve or enhance the production of a specific antioxidant, but this may limit the production of other antioxidants. Funding: This research was funded by the Instituto Politécnico Nacional, grant number: 1947 Proyecto de Investigación 2020. This publication was supported by Comisión de Operación y Fomento de Actividades Académicas (COFAA) del Instituto Politécnico Nacional.