Enhancement of Astaxanthin and Fatty Acid Production in Haematococcus pluvialis Using Strigolactone

Abstract

Improving the production rate of high-value nutraceutical compounds, such as astaxanthin and polyunsaturated fatty acids (PUFAs), is important for the commercialization of Haematococcus pluvialis biorefineries. Here, the effects of a phytohormone, strigolactone analog rac-GR24, on cell growth and astaxanthin and fatty acid biosynthesis in H. pluvialis were investigated. Four concentrations (2, 4, 6, and 8 µM) of rac-GR24 were initially added during 30 days of photoautotrophic cultivation. The addition of rac-GR24 improved cell number density and chlorophyll concentration in H. pluvialis cultures compared to the control; the optimal concentration was 8 µM. Despite a slightly reduced astaxanthin content of 30-d-old cyst cells, the astaxanthin production (26.1 ± 1.7 mg/L) improved by 21% compared to the rac-GR24-free control (21.6 ± 1.5 mg/L), owing to improved biomass production. Notably, at the highest dosage of 8 µM rac-GR24, the total fatty acid content of the treated H. pluvialis cells (899.8 pg/cell) was higher than that of the untreated cells (762.5 pg/cell), resulting in a significant increase in the total fatty acid production (361.6 ± 48.0 mg/L; 61% improvement over the control). The ratio of PUFAs, such as linoleic (C18:2) and linolenic (C18:3) acids, among total fatty acids was high (41.5–44.6% w/w) regardless of the rac-GR24 dose.

1. Introduction

Microalgae have long been utilized as industrial microorganisms to produce high-value nutraceutical biocompounds, such as astaxanthin and polyunsaturated fatty acids (PUFAs) [1]. Recently, astaxanthin (3,3-dihydroxy-β-carotene-4,4-dione, Figure 1a), a secondary ketocarotenoid pigment, has received considerable attention in various bioindustries, including food supplements, cosmetics, and aquaculture [2]. The antioxidant activity of astaxanthin is significantly higher than that of β-carotene and vitamin E. This pigment has various therapeutic effects, such as immunomodulation, anti-aging activity, anti-cancer potential, and cardiovascular protection [3].

Haematococcus pluvialis, a freshwater unicellular green microalga, is considered one of the best sources of astaxanthin (~5% of dry weight) [1,4]. Algal astaxanthin is usually synthesized as a self-defense mechanism to protect the organism from oxidative stress. In H. pluvialis, astaxanthin biosynthesis is induced during the transformation of vegetative cells into dormant cysts under unfavorable culture conditions [5]. Astaxanthin biomolecules accumulate inside neutral lipid bodies forming ester bonds with long-chain fatty acids, enabled by closely interconnected metabolic networks of lipogenesis and carotenogenesis [6]. To improve the astaxanthin productivity of photosynthetic H. pluvialis cultures, various stress-inducing strategies, such as nitrogen starvation [7], strong light irradiance [8], high salinity [9], high temperature [10], mechanical stress [11], electrical treatment [12], and aminoclay nanoparticle addition [2] have been applied. However, these approaches are time-consuming, require high energy input and large amounts of chemicals; additionally, they may substantially inhibit cell growth. Therefore, it is necessary to develop an efficient astaxanthin production method applying environmentally friendly large-scale biorefinement of H. pluvialis.

Strigolactone, a carotenoid-derived phytohormone, functions as an effective chemical messenger that regulates cellular activity in plants in response to nutrient availability and environmental changes [13]. Strigolactone also plays an important role in shoot branching, leaf senescence, and symbiosis with microorganisms [14]. Recently, Shen et al. [13] have reported that the addition of strigolactone analog rac-GR24 (0.001–1 μM) (see Figure 1b for the chemical structure) enhances the photosynthetic growth rate of Chlorella vulgaris FACHB-8 by increasing chlorophyll a content compared to the control. Interestingly, rac-GR24 (0.2–5 μM) also promotes lipid accumulation in Monoraphidium sp. QLY-1 when CaCl₂ supplementation [14] or nitrogen deficiency [15] strategies are combined. These results suggest that, although cell growth and lipid biosynthesis are generally inversely related, rac-GR24 may help improve overall lipid productivity in other algal species if applied carefully.

Notably, astaxanthin and lipid biosynthetic pathways in H. pluvialis are closely linked [1,4]. In addition, strigolactone is expected to enhance the germination rate of dormant cysts of H. pluvialis, similar to that observed in plant seeds [16]. In this study, we aimed to investigate the technical feasibility of using rac-GR24 to improve astaxanthin and fatty acid production in photosynthetic H. pluvialis cultures.

2. Materials and Methods

2.1. Chemicals

Strigolactone analog rac-GR24 (purity ≥ 98%) was purchased from Chiralix B.V. (Nijmegen, The Netherlands). The rac-GR24 stock solution was prepared in acetone at a concentration of 10 mM and used for H. pluvialis cultivation. Astaxanthin and fatty acid methyl ester (FAME) (Mix RM3, Mix RM5, GLC50, and GLC70) standards were acquired from Dr. Ehrenstorfer GmbH (Augsburg, Germany) and Sigma Aldrich (St. Louis, MO, USA), respectively. Distilled water, organic solvents, and chemicals used in this study were of analytical grade and obtained from Junsei (Tokyo, Japan) and Sigma Aldrich.

2.2. Microalga and Photosynthetic Cultivation

H. pluvialis NIES-144, obtained from the National Institute for Environmental Studies (NIES, University of Tokyo, Tokyo, Japan), was grown under photosynthetic conditions [17,18]. NIES-C medium was used to culture H. pluvialis with the following composition (per liter): 0.15 g Ca(NO₃)₂·4H₂O, 0.10 g KNO₃, 0.05 g β-glycerophosphate disodium salt hydrate, 0.04 g MgSO₄·7H₂O, 0.05 g tris(hydroxymethyl)aminomethane, 0.01 mg thiamine, 0.10 μg biotin, 0.10 μg vitamin B₁₂, and 3.00 mL PIV metal solution. One liter of PIV metal solution consisted of 1.00 g Na₂EDTA, 196.00 mg FeCl₃·6H₂O, 36.00 mg MnCl₂·4H₂O, 22.00 mg ZnSO₄·7H₂O, 4.00 mg CoCl₂·6H₂O, and 2.50 mg Na₂MoO₄·2H₂O. The NIES-C medium was adjusted to pH 7.5 and filtered using a 0.2 μm membrane filter (mixed cellulose ester, 47 mm diameter; Toyo Roshi Kaisha Ltd., Tokyo, Japan). The algal seed culture was maintained in a 300 mL Erlenmeyer flask (working volume, 200 mL) with a porous silicon stopper in a shaking incubator (25 °C, 150 rpm, and a continuous light of 80 μmol/m²/s; ISF-7100RF, Jeio Tech, Daejeon, Korea).

The primary photosynthetic culture of H. pluvialis with rac-GR24 was maintained for 30 d under the same seed culture conditions. The 30-d-grown seed culture was collected and inoculated to obtain an optical density (OD) of 0.1 at 680 nm (UV/Vis spectrophotometer, Optizen 3220 UV; Mecasys Co., Daejeon, Korea). Rac-GR24 was initially added at four different concentrations (2, 4, 6, and 8 µM) to the flasks, based on previous reports on the green microalgae C. vulgaris FACHB-8 [13] and M. sp. QLY-1 [14].

2.3. Astaxanthin and Fatty Acid Quantification

After 30 d of cultivation, H. pluvialis cyst cells were harvested by centrifugation at 3000 rpm for 10 min (Combi R515; Hanil Science Co., Daejeon, Korea), double-washed with distilled water, freeze-dried for 1 d (FD8508; IlShin BioBase Co. Ltd., Dongducheon, Korea), and stored at −22 °C until further analysis.

Approximately 2 mg of freeze-dried algal cells were added to a 2 mL bead-beating tube containing 1.0 g micro-beads (1.5 mm diameter; Daihan Scientific, Gangwon, Korea) and 1 mL of dichloromethane and methanol mixture (1:1, v/v) at 0.025 M NaOH to extract astaxanthin. The H. pluvialis cells were mechanically disrupted using a FastPrep-24 bead-beater (6 m/s for 30 s and 3 cycles; MP Biomedicals, Irvine, CA, USA). The astaxanthin extract was further incubated in the dark for 2 h at 4 °C for saponification. Astaxanthin content was measured using high-performance liquid chromatography (HPLC, Agilent 1260 Infinity system, Agilent Technologies, Santa Clara, CA, USA) equipped with a diode-array detector and a YMC carotenoid column (4.6 mm (ID] × 250 mm [length], 5 μm [particle size]; YMC Inc., Kyoto, Japan). The detailed HPLC conditions are available in our previous publications [2,19].

Fatty acid content was estimated as the amount of FAME through a direct transesterification reaction, followed by gas chromatography (GC) analysis according to a previous protocol [17]. Briefly, 10 mg of freeze-dried algal cells were placed in a 12 mL Pyrex glass tube containing 2 mL of chloroform and methanol mixture (2:1, v/v), and vigorously mixed for 10 min to extract lipids. For FAME analysis, 1 mL methanol, 300 µL sulfuric acid (H₂SO₄) (95%), and 1 mL chloroform containing heptadecanoate (17:0) as an internal standard was added to the tube, mixed by vertexing for 5 min (Vortex V3 S0A0; IKA, Staufen, Germany), and incubated at 100 °C for 10 min using a heating block (HS R200; Humas, Daejeon, Korea). The reaction tube was cooled to room temperature, supplemented with 1 mL distilled water, and vortexed for 5 min. After centrifugation at 3000 rpm for 10 min, the lipid extract fraction in the bottom layer of the reaction mixture was filtered and analyzed using a GC) equipped with a flame ionization detector and a capillary HP-INNOWax column (0.32 mm [ID] × 30 m [length], 0.5 µm [film thickness]; Agilent Technologies).

2.4. Other Analyses

The cell number density of H. pluvialis was determined using an improved Neubauer hemocytometer (DHC-N01-5; INCYTO, Chungnam, Korea). Morphological characteristics and cell diameter were analyzed using an Axiolab bright-field microscope equipped with a digital camera and Zen lite 2012 software (Carl Zeiss, Jena, Germany). Light intensity and pH were measured using an LI-250A quantum photometer (LI-COR Inc., Lincoln, NE, USA) and pH meter (HM-30R; TOADKK, Tokyo, Japan), respectively.

Chlorophyll concentration of H. pluvialis cultures was monitored based on solvent extraction and spectrophotometric assays according to a previous report [2]. Briefly, 1 mL of algal solution from the flask culture was collected in a bead-beating tube and centrifuged for 10 min at 10,000 rpm (Legend Micro 17R; Thermo Fisher Scientific, Waltham, MA, USA). After discarding the supernatant, 1.0 g micro-beads were added and resuspended in 1 mL acetone. The algal cells were mechanically disrupted using a bead beater (6 m/s for 30 s, 3 times). Chlorophyll a and b concentrations were estimated using the following equations (mg/L) based on the OD values at 644.8 and 661.6 nm [20]:

$$\mathrm{Chlorophyll} a=11.24\times {\mathrm{OD}}_{661.6}-2.04\times {\mathrm{OD}}_{644.8}$$

$$\mathrm{Chlorophyll} b=20.13\times {\mathrm{OD}}_{644.8}-4.19\times {\mathrm{OD}}_{661.6}$$

$$\mathrm{Chlorophyll} a \mathrm{and} b=\mathrm{Chlorophyll} a+\mathrm{Chlorophyll} b$$

2.5. Statistical Analysis

Plotting and statistical data analysis were performed using SigmaPlot 14.0 (Systat Software Inc., San Jose, CA, USA) and SPSS Statistics 17.0 (IBM Co., Chicago, IL, USA), respectively. The experimental results are expressed as the mean ± standard deviation derived from independent duplicate algal cultivation. Statistical significance (p < 0.05) was determined using a t-test for significance level.

3. Results and Discussion

3.1. Growth and Morphology of H. pluvailis with rac-GR24

Figure 2 shows the changes in the total cell number density, optical shape, and relative morphological distribution of H. pluvialis during 30-d photosynthetic cultivation. To enhance cell growth and astaxanthin and fatty acid production in H. pluvialis, strigolactone analog rac-GR24 was initially added at 2.0–8.0 μM. Overall, the cell number density of the algal cultures tended to increase almost proportionally with increasing rac-GR24 dosage in the tested range. For the highest rac-GR24 dosage (8.0 μM), a considerably higher cell number density trend (~429.4 × 10³ cells/mL) was observed during the photosynthetic culture, which was approximately 25% higher than that observed in the rac-GR24-free control (~342.5 × 10³ cells/mL). Regardless of rac-GR24 addition, the cell number densities of all H. pluvialis cultures increased almost linearly with time for the initial 12 days. However, as the nitrogen source was depleted, the growth rate of H. pluvialis decreased, and all algal cultures soon reached stationary growth phases with astaxanthin induction [2]. It should be noted that the difference in cell number density observed at rac-GR24 concentrations of 2–8 μM appeared to be much smaller than between them and zero (control). This implies that, although not investigated in this study, there may be a threshold value below 2 μM. This could be an interesting topic for further investigation.

The optical appearance of H. pluvialis cells during the photosynthetic life cycle (Figure 2b) can be divided into five different cell types, such as red cyst cells, germinating cells, biflagellate cells, green palmella cells, and brownish-green/brown palmella cells, similar to previous observations [1,12]. When inoculated into fresh medium, red cyst cells started to germinate rapidly. The germinating cells formed sporangia and proliferated internal daughter cells. The cell membrane then broke to open, releasing motile biflagellate cells. With cell aging, the motile biflagellate cells lose their flagella and transform into vegetative non-motile green palmella. The palmella cells reproduced asexually with 2, 4, 8, 16, or 32 daughter cells. When exposed to adverse conditions, such as nutrient limitation and excessive light irradiation, astaxanthin biosynthesis is induced in the palmella cells as an adaptive defense event [1,21]. Astaxanthin accumulation was observed in the center and spread to the periphery, gradually changing the entire cell color from green to brownish-green and brown in turn. Eventually, they were transformed into a dormant cyst with a rigid cell wall structure that was rich in astaxanthin and exhibited a bright red color.

Overall, the morphological distribution patterns of H. pluvialis cell types during 30 d cultivation were similar regardless of rac-GR24 concentration (Figure 2c). However, it should be noted that the rac-GR24-treated groups (8.6 ± 2.0%) showed slightly higher germination rates during the first two days than the control (7.9 ± 0.8%). This might have contributed to the improvement in the cell number density of the former (Figure 2a). Interestingly, after 30 d cultivation, the cell size of H. pluvialis showed a tendency to slightly increase as the rac-GR24 concentration increased from 0 to 8 μM (Figure 3). Strigolactone analog rac-GR24 promotes the growth of microorganisms and plants by alleviating reactive oxygen species (ROS) levels and excess oxidative stress [14]. Rac-GR24 improves the biomass productivity of C. vulgaris FACHB-8 [13] and M. sp. QLY-1 [22] and stimulates mitosis and growth of arbuscular mycorrhizal fungi Gigaspora rosea DAOM 194757 by boosting energy metabolism [23].

3.2. Chlorophyll a and b Concentrations

The photosynthetic activity of H. pluvialis cells during the 30-d cultivation under different rac-GR24 concentrations (2.0–8.0 μM) was estimated based on the concentrations of chlorophyll a and b, the main light-harvesting pigments [24] (Figure 4). Similar to the cell number density results (Figure 2), the chlorophyll a and b concentrations of the rac-GR24-treated algal cultures were higher than those of the untreated control and also showed an increasing trend with increasing rac-GR24 dosage. The highest chlorophyll a and b concentration (19.6 ± 0.9 mg/L) at the 8 µM rac-GR24-added culture significantly improved by 50.3% compared to the control (13.1 ± 0.1 mg/L). The enhanced cell number density of the rac-GR24-treated algal cells might be mainly due to the improved chlorophyll a and b concentrations along with the earlier germination-promoting effect (Figure 2c). Shen et al. [13] observed increased chlorophyll a content in C. vulgaris FACHB-8 at 0.1 μM rac-GR24. Dong et al. [25] observed the stimulating effect of rac-GR24 on carbonic anhydrase activity during microalgae-fungi-bacteria symbiont cocultures, such as C. vulgaris-Ganoderma lucidum-endophytic bacteria. Similarly, rac-GR24 enhanced the chlorophyll content and photosynthesis rate of plants, such as Vitis vinifera L. [26] and Brassica napus L. [27], under stressful drought and salinity conditions, respectively.

In Figure 4, the chlorophyll a and b concentrations of all H. pluvialis cultures showed a decreasing trend after peaking around day 18, similar to the cell number density results (Figure 2a). This may be related to the physiological change of H. pluvialis cells from chlorophyll-synthesizing vegetative growth to the dormant encystment with astaxanthin induction as the nitrogen nutrient was depleted (see Figure 2c). In the absence of additional photosynthetic growth, chlorophyll pigments can be degraded and reallocated to support the metabolic activity of algal cells exposed to adverse culture conditions. Solovchenko [28] reported that during the photosynthetic cultures of H. pluvialis, Chlamydomonas reinhardtii, and C. zofingiensis, down-regulation of chlorophyll synthesis was associated with subsequent upregulation of astaxanthin and lipid synthesis.

3.3. Astaxanthin and Fatty Acid Production

Figure 5 shows the cellular content and volumetric production of astaxanthin and total fatty acids in 30-d-grown H. pluvialis cells at different doses of rac-GR24 (2.0–8.0 μM). Contrary to the results of the cell growth promotion (Figure 2a), the astaxanthin contents of the rac-GR24-treated cells (60.1–65.0 pg/cell) were slightly lower than that of the control (73.3 pg/cell). This indicates that rac-GR24 does not promote astaxanthin biosynthesis in H. pluvialis cells under the present photoautotrophic culture conditions. Cell growth rate and astaxanthin accumulation in H. pluvialis generally show opposite trends under photosynthetic conditions [2,28]. However, it should be noted that at the highest dosage of 8 µM rac-GR24, the volumetric astaxanthin production (26.1 ± 1.7 mg/L) considerably improved by 21% compared to the control (21.6 ± 1.5 mg/L) due to the improvement of cell number density. Oxidative stress, such as nutrient depletion, electric stress, and strong light irradiation, induces the generation of toxic ROS, which severely damages the cell membrane, DNA, and proteins of algal cells. In terms of cellular defense mechanisms, ROS also serves as an important signal that triggers the metabolic synthesis of various antioxidant biomolecules, such as astaxanthin [29]. This suggests that rac-GR24 may modulate the cellular antioxidant system to eliminate excess ROS while simultaneously interfering with the astaxanthin-induced event. However, Hu et al. [30] reported an enhancement of astaxanthin production in H. pluvialis 192.80 by combining an organic carbon substrate acetate with another phytohormone, salicylic acid, under photoheterotrophic conditions. Ding et al. [31] improved astaxanthin content by 2.36-fold in H. pluvialis LUGU using 10 μM melatonin (N-acetyl-5-methoxytryptamine) under photoautotrophic conditions. The effects of phytohormonal types and nutritional modes on H. pluvialis may be subject to further investigation.

Interestingly, the effect of rac-GR24 on the accumulation of total fatty acids in H. pluvialis cells was different from that of astaxanthin (Figure 5). At the lowest dosage of 2 µM rac-GR24, the fatty acid content of H. pluvialis was lower as 628.2 pg/cell compared to the untreated control (762.5 pg/cell), similar to that of astaxanthin. However, at concentrations above 2 µM, with increasing rac-GR24 dose, the algal fatty acid content showed an increasing trend. Notably, the highest dose of 8 µM rac-GR24 resulted in a considerable increase in the total fatty acid content (899.8 ± 119.5 pg/cell) than the control (762.5 ± 108.5 pg/cell). Furthermore, due to the enhanced cell number density (Figure 2), the volumetric total fatty acid production increased almost proportionally with increasing the rac-GR24 dose in the range of 2–8 μM, obtaining a 1.6-fold improvement (361.6 ± 48.0 mg/L) at 8 μM compared to the untreated control (224.5 ± 31.9 mg/L). This suggests that rac-GR24 can enhance lipid production in the photosynthetic H. pluvialis culture.

Enhancement of algal lipid biosynthesis using several phytochemicals has been reported. Song et al. [14] observed that 1 μM rac-GR24 enhanced the lipid productivity of M. sp. QLY-1 by 54.6% with CaCl₂ supplementation compared to the untreated control. Endogenous NO and Ca²⁺ levels were triggered by adding rac-GR24. Chemical genetic data demonstrated that Ca²⁺ accumulation induced by rac-GR24 stimulates lipid accumulation by regulating the transcription levels of lipid biosynthesis-related genes and NO signaling. They also reported a 1.3-fold improvement in lipid productivity with the same rac-GR24 dosage under nitrogen-deficient conditions [15]. Compared to the control group, melatonin improved the lipid content of H. pluvialis LUGU by 49.5% under photoautotrophic conditions [32], and salicylic acid increased the total fatty acid content of H. pluvialis 192.80 by 36.0% under photoheterotrophic conditions [30]. The combination of melatonin and nitrogen deficiency improved the lipid content in M. sp. QLY-1 by 1.22-fold [33]. Indole-3-propionic acid promoted growth and lipid productivity in C. pyrenoidosa FACHB5 and Scenedesmus quadricauda FACHB506 [34]. This suggests that the effects of phytohormones on algal lipids are highly dependent on species and culture conditions, which may be an interesting topic for further research.

Although the complex metabolic networks have not been clearly elucidated, the biosynthesis of fatty acids and neutral lipids in H. pluvialis is believed to be closely related to the biosynthesis and storage of astaxanthin [5,6]. To understand the different accumulation patterns between astaxanthin and total fatty acids observed in this study (Figure 5), detailed genetic and biochemical investigations are required. The effect of higher rac-GR24 concentration above 8 μM on astaxanthin and lipid biosynthesis may be also the subject of further investigation.

H. pluvialis is regarded as a fatty acid-rich algal species available for commercial use in food and nutritional products [1]. PUFAs have gained considerable public recognition in terms of their therapeutic potential, such as reduced risk of cardiovascular disease.

Table 1. Fatty acid composition of Haematococcus pluvialis after 30 d cultivation under different rac-GR24 concentrations (2.0–8.0 μM).

Fatty Acid Composition (%)
rac-GR24 Concentration (µM)		0	2	4	6	8
Myristate	(C14:0)	0.2 ± 0.1	0.3 ± 0.0	0.4 ± 0.0	0.4 ± 0.0	0.3 ± 0.0
Palmitate	(C16:0)	35.2 ± 2.0	31.7 ± 1.3	30.5 ± 0.8	27.6 ± 1.2	27.2 ± 1.0
Palmitoleate	(C16:1)	0.1 ± 0.2	0.3 ± 0.2	0.5 ± 0.0	0.5 ± 0.0	0.5 ± 0.0
Oleate	(C18:1n9c)	19.1 ± 1.1	18.2 ± 0.8	16.4 ± 0.6	16.1 ± 0.3	17.3 ± 0.1
Linoleate	(C18:2n6c)	34.7 ± 1.1	33.0 ± 0.1	34.7 ± 0.3	34.3 ± 1.2	34.1 ± 0.4
γ-Linolenate	(C18:3n2c)	0.5 ± 0.7	1.6 ± 0.1	1.7 ± 0.0	1.81 ± 0.0	1.9 ± 0.0
Linolenate	(C18:3n3c)	5.1 ± 0.5	6.9 ± 0.2	7.0 ± 0.5	7.7 ± 0.3	8.6 ± 0.4
Others		5.0 ± 1.8	8.0 ± 1.6	8.8 ± 1.1	11.7 ± 3.0	10.1 ± 1.0
Saturated fatty acids		35.4 ± 1.9	32.0 ± 1.3	30.9 ± 0.8	27.9 ± 1.3	27.5 ± 1.0
Monounsaturated fatty acids		19.2 ± 1.1	18.5 ± 0.6	16.9 ± 0.6	16.5 ± 0.4	17.8 ± 0.1
Polyunsaturated fatty acids (PUFAs)		40.4 ± 0.9	41.5 ± 0.3	43.4 ± 0.2	43.8 ± 1.4	44.6 ± 0.2
Others		5.0 ± 1.8	8.0 ± 1.6	8.8 ± 1.1	11.7 ± 3.0	10.1 ± 1.0
Total		100	100	100	100	100

shows the fatty acid profiles of mature H. pluvialis cyst cells after the 30 d cultivation with different rac-GR24 concentrations (2.0–8.0 μM). Overall, the addition of rac-GR24 increased the composition of palmitoleic (C16:1), γ-linolenic (C18:3n2c), and linolenic (C18:3n3c) acids in the total fatty acids, whereas it decreased the ratio of palmitic (C16:0) and oleic (C16:1) acids. Accordingly, the PUFA content (41.5–44.6%) of the rac-GR24-treated H. pluvialis cells was slightly higher than that of the control (40.4%). It should be noted that the total fatty acid production was significantly improved by ~60% (Figure 5), although the change in cellular PUFA content was small. Similarly, Cui et al. [32] reported that the combination of melatonin and CaCl₂ promoted the PUFA ratio among the total fatty acids in H. pluvialis LUGU. Salama et al. [35] observed the promotion of PUFA biosynthesis in C. mexicana GU732420 and S. obliquus HM103382 in the presence of the phytohormone indole-3-acetic acid.

4. Conclusions

Addition of 2–8 μM rac-GR24 improved the cell number density of H. pluvialis during 30-d photosynthetic cultivation. The improvement in cell growth was mainly attributed to an enhanced physiological germination rate and higher chlorophyll a and b concentrations. After 30-d of cultivation, astaxanthin and total fatty acid contents of the rac-GR24-treated cells were lower or comparable to those of the untreated controls at the doses evaluated. Nevertheless, at the highest dose of 8 μM rac-GR24, thanks to the increased cell number density, both astaxanthin and total fatty acid production significantly improved to 26.1 mg/L and 361.6 mg/L, respectively, which were 21% and 60% higher than those of the controls. The proportion of polyunsaturated fatty acids among the total fatty acids was high (41.5–44.6%) in the rac-GR24-treated cells. These results indicate that rac-GR24 can promote both astaxanthin and PUFA production in photosynthetic H. pluvialis cultures.