The genus Haematococcus, which comprises unicellular green freshwater microalgae, is recognized as relatively diverse with several species. However, just one species has been formally identified for a long time. This species was initially assigned two names, Haematococcus pluvialis and Haematococcus lacustris, but later determined to be synonymous. Currently, the species is now known as H. lacustris based on the study of Nakada and Ota [1].

H. lacustris, is regarded as the best natural source of astaxanthin, a potent antioxidant and red pigment with extensive applications in the functional food, pharmaceutical, cosmetic, and aquaculture industries [2]. The antioxidant activity of astaxanthin is 65 times higher than that of vitamin C, 54 times that of β-carotene, 100 times that of α-tocopherol and vitamin E, and 10 times that of zeaxanthin and lutein [2]. Astaxanthin accumulates in Haematococcus cells when the green algal cells are transformed into red cystic cells under stress [3]. Several environmental factors affect astaxanthin biosynthesis; high light intensity (160 μmol m⁻² s⁻¹) [4], high temperature (28°C) [5], nitrogen deficiency (0.0mM nitrate) [6], or presence of phosphate (41 mg/L) [7] induce astaxanthin synthesis. Acetate as a carbon source (5 mM) [8] and NaCl at a concentration of 2.2 mM [9] also affect astaxanthin content.

Among the various stressors applied to Haematococcus, light is the most important factor that increases astaxanthin accumulation in H. lacustris [10]. Several studies have considered light intensity as a major induction factor; however, it can also significantly inhibit photosynthesis or damage cells due to photo-oxidation [11,12]. Moreover, light intensity has a greater effect on astaxanthin production than the C/N ratio during photoautotrophic induction [3]. Therefore, light intensity can be used to directly control algal growth and metabolism.

As light intensity plays an important role in inducing astaxanthin synthesis in Haematococcus, it is important to establish an efficient optical system to produce astaxanthin using LED instead of conventional light sources. LEDs exhibit a narrower emission spectrum, higher conversion efficiency, and less heat emission than conventional fluorescent lamps [13]. Additionally, they are suitable for photobioreactors because of their low power consumption, small chip size, and long shelf-life [14,15]. Owing to these advantages, LEDs are the preferred light source for microalgal cultures. The main wavelengths of LEDs applicable to microalgal biotechnology are blue, green, and red, all of which have distinct effects on microalgal biology. Among them, red and blue LEDs are most effective in inducing photosynthesis.

Although LEDs are the most efficient light sources for photosynthesis [16], their effect on the two physiological stages of H. lacustris vary. As red light induces cell division and blue light stimulates cell maturation, red lights are used for enhancing growth rate and blue lights are used during the cystic phase of astaxanthin accumulation [13,17]. Blue lights have considerable advantages over red or white lights in astaxanthin production under monochromatic light illumination; a higher amount of astaxanthin accumulates in the presence of blue light and mixed light of other wavelengths [18]. Moreover, mixed LED lighting with red (660 nm) and blue (465 nm) wavelengths is more suitable for microalgal growth than lights of other wavelengths, and it also accelerates photosynthesis [19]. Although the spectral distribution and mechanisms that enhance cyst formation and astaxanthin accumulation in H. lacustris have been elucidated [20], studies on the effects of red and blue spectral distributions on the growth of H. lacustris are limited. Therefore, it is, necessary to determine the specific LED wavelengths for the optimal manipulation of metabolism of H. lacustris to improve biomass and astaxanthin production.

In this study, we investigated the growth of H. lacustris under different light intensities and mixing ratios of red and blue LEDs. Our study may provide basic data for future research on the complex and condition-dependent photosynthetic reactions of H. lacustris, as well as mass-culture of microalgae by optimizing the light source and intensity for H. lacustris growth.

2.1. Strain and culture conditions

Two strains of H. lacustris, UTEX 2505 and UTEX 16 (Fig. 1), were obtained from the Culture Collection of Algae at the University of Texas, Austin, TX, USA. All cultures were prepared using MES-Volvox medium (UTEX Culture Collection of Algae) composed of 0.118 g Ca(NO3)2·4H₂O, 0.04 g MgSO4·7H₂O, 0.05 g sodium glycerophosphate, 0.05 g KCl, 0.0267 g NH4Cl, 0.0045 g disodium-EDTA, 0.000582 g FeCl₃·6H₂O, 0.000246 g MnCl₂·4H₂O, 0.00003 g ZnCl₂·6H₂O, 0.000012 g CoCl₂·6H₂O, 0.000024 g Na2MoO4·2H₂O, 0.000025 g biotin, 0.000135 g vitamin B12 (cyanocobalamin), 0.012 g HEPES buffer (pH 7.8), and 1.95 g MES per liter of distilled water. The seed culture was grown at 20°C ± 1°C and irradiated with a white fluorescent lamp at a light intensity of 60 μmol m⁻² s⁻¹ for 24 h. Each H. lacustius was maintained in MES-Volvox medium for 3days.

Figure 1. Micrographs of Haematococcus lacustris UTEX 16 (a) and H. lacustris UTEX 2505 (b) obtained using light microscopy. Scale bars = 20 μm

2.2. Red, green, blue (RGB)-LED light intensity controller

The LED light multi-controller device comprised 40 test tubes that could be stirred in separate compartments, and the light intensity of the RGB LEDs could be controlled using a personal computer (PC). A calibration curve for controlling the light intensity through a PC by converting the electrical adjustment value to a light intensity value is shown in Fig. 2.

Figure 2. Calibration curve and regression equation for each wavelength of three-wavelength LED, adjustment value, and light quantity according to the depth of the test tube.

Light intensity was determined by measuring the distance between the light source and sample in a test tube, using a specially manufactured aluminum tube and an inserted light sensor. The regression equation for the red wavelength was the exponential equation (y = ax^b), and the blue and green wavelengths were regressed using the linear equation (y = ax + b). The equations were determined to be reliable with an R² value of 0.999 – 1.000.

The optimal growth tester was designed by selecting the RS485 communication for PC control, with the software for light control being directly produced. In the software configuration, identification numbers (IDs) from 1 to 40 were assigned to the port setting, and the LED device of each test tube and the light intensity of the three wavelengths was adjusted accordingly.

2.3. Light conditions

A fluorescent lamp and red (620-630 nm), green (520-530 nm), and blue (455-465 nm) LEDs were used for the culture. The intensities of the fluorescent lamp light and the incident light on the cultures at the distance from the lamps were measured using a light meter (LI-250A; Li-Cor, Lincoln, NE, USA) with a quantum sensor (LI-250A; Li-Cor, Lincoln, NE, USA). The light intensity of LEDs was controlled using the RGB-LED light intensity controller (Fig. 3).

Figure 3. Red, green, and blue (RGB)-LED light multi-controller.

2.3.1. Monochromatic light intensity

Fluorescent lamps and RGB LEDs were used as light sources for the experiments to investigate the effects of monochromatic light intensity on growth. The light intensities with seven different irradiations are shown in Table 1.

Table 1 Monochromatic light intensity used in Haematococcus lacustris growth experiments

	Exp 1	Exp 2	Exp 3	Exp 4	Exp 5	Exp 6	Exp 7
Light intensity (μmol m−2 s−1)	0	10	20	50	80	100	200

2.3.2. Optimal light intensity

Red and blue LEDs were used as light sources for the experiments to optimize the light intensity for growth. The red and blue LEDs were mixed at a 1:1 ratio. The intensities of the light sources with eight different irradiation experiments are presented in Table 2.

Table 2 Intensities of red and blue lights used in Haematococcus lacustris growth experiments

	Exp 1	Exp 2	Exp 3	Exp 4	Exp 5	Exp 6	Exp 7	Exp 8
Total light intensity (μmol m−2 s−1)	0	10	20	50	80	100	200	300
Red LED (μmol m−2 s−1)	0	5	10	25	40	50	100	150
Blue LED (μmol m−2 s−1)	0	5	10	25	40	50	100	150

2.3.3. Optimal red:blue LED ratio

To determine the optimal light ratio using a mixture of red and blue LEDs, the cultures were illuminated at the same total light intensity (100 μmol m⁻² s⁻¹) with nine different combinations red:blue LED ratios. The details are presented in Table 3.

Table 3 Mixing ratios of red and blue lights used in Haematococcus lacustris growth experiments

	Exp 1	Exp 2	Exp 3	Exp 4	Exp 5	Exp 6	Exp 7	Exp 8	Exp 9
Total light intensity (μmol m−2 s−1)	100	100	100	100	100	100	100	100	100
Red LED (μmol m−2 s−1)	83.3	80	75	66.7	50	33.3	25	20	16.7
Blue LED (μmol m−2 s−1)	16.7	20	25	33.3	50	66.7	75	80	83.3
Ratio (red: blue)	5:1	4:1	3:1	2:1	1:1	1:2	1:3	1:4	1:5

2.4. Growth measurements

The strains were maintained in MES-Volvox medium under 60 μmol m⁻² s⁻¹ cool-white, fluorescent illumination with 24-h light. The volume of the culture was maintained at 20 mL using a test tube, initial cell concentration was 100,000 cells mL⁻¹, and temperature was maintained at 20°C ± 1°C; the cells were cultured for 10 d. All experiments were performed in triplicate. The light intensity was set using an RGB-LED light intensity controller, and the experiments were conducted in a 24-h light cycle. The culture in the test tube was mixed every 48 h, and 3 mL of the cell suspension was sampled and fixed with Lugol’s solution. The number of cells was determined by direct counting under a microscope (BX 53; Olympus, Tokyo, Japan). UTEX 2505 and UTEX 16 cells were counted using Sedgewick–Rafter Chambers (SRCs). The conditions of the cells were assessed under a dissecting microscope (BX 53; Olympus) before obtaining subsamples.

3.1. Effect of light intensity (monochromatic light) on the growth of H. lacustris

Significant differences were observed in the growth of UTEX 2505 after 10 d of culture under monochromatic light intensities of 0-100 μmol m⁻² s⁻¹ (Fig. 4). The cell concentration reached a maximum value of 944,000 cells mL–1 under blue LED within 10 d of culture, whereas the cell concentration increased to approximately 674,000 cells mL⁻¹ under green LED and 413,000 cells mL⁻¹ under the fluorescent lamp at the end of the 10-d incubation period. The cell concentration was significantly lower under red LED than under blue LED of the same light intensity for UTEX 2505.

Figure 4. Effect of different monochromatic light intensities (μmol m⁻² s⁻¹) on the concentration of Haematococcus lacustris (UTEX 2505) cells. (a) Red LED, (b) green LED, (c) blue LED, and (d) fluorescent lamp.

The concentration of UTEX 16 cells was the maximum (653,000 cells mL⁻¹) under blue LED and minimum under red LED, at a light intensity of 100 μmol m⁻² s⁻¹ for 10 d. During cultivation periods, UTEX 16 cell concentration increased by 6.5 times compared with the initial cell concentration under blue LED at 100 μmol m⁻² s⁻¹ (Fig. 5), representing an 87% increase in cell growth compared with that under red LED of the same light intensity after the 10 d culture period.

Figure 5. Effect of different monochromatic light intensities (μmol m⁻² s⁻¹) on the concentration of Haematococcus lacustris (UTEX 16) cells. (a) Red LED, (b) green LED, and (c) blue LED, and (d) fluorescent lamp.

3.2. Effects of light intensity (mixed red and blue LED) on the growth of H. lacustris

The growth curves for UTEX 2505 and UTEX 16 under different mixed red and blue LED (1:1) light intensities are presented in Fig. 6. All data points in Fig. 6 are the average of triplicate tests. The optimal light intensity for the best cell growth of both strains was 100 μmol m⁻² s⁻¹. Cell growth under lower (0–80 μmol m⁻² s⁻¹) or higher light intensities (200–300 μmol m⁻² s⁻¹) did not conform to that observed under optimal light intensity (100 μmol m⁻² s⁻¹).

Figure 6. Effect of mixed red and blue LED (1:1) light intensities (μmol m⁻² s⁻¹) on the concentration of Haematococcus lacustris UTEX 16 (a) and H. lacustris UTEX 2505 (b).

As shown in Fig. 6, from day 4 of culture, significant differences in the growth of UTEX 2505 cells were observed under different mixed light intensities. The growth of cells was the maximum (376,800 cells mL⁻¹) at a light intensity of 100 μmol m⁻² s⁻¹, and it was slightly less at 200 and 300 μmol m⁻² s⁻¹. At light intensities of 0, 10, and 20 μmol m⁻² s⁻¹, the cells did not grow instead the initial cell concentration tended to continuously decrease during the experiment.

Significant differences in the growth of UTEX 16 cells under different light intensities were observed from day 6 of culture (Fig. 6). The concentration of UTEX 16 cells reached a maximum value of 358,800 cells mL⁻¹ at a light intensity of 100 μmol m⁻² s⁻¹, and it was slightly less at 200 and 300 μmol m⁻² s⁻¹. At light intensities of 0 and 10 μmol m⁻² s⁻¹, the cells did not grow and the cell concentrations tended to continuously decrease.

The maximum growth of both H. lacustris strains was observed at a light intensity of 100 μmol m⁻² s⁻¹. Moreover, the cell concentrations increased to a maximum value at 100 μmol m⁻² s⁻¹, but they were less at 200 and 300 μmol m⁻² s⁻¹, with a greater growth reduction at a higher light intensity. This trend indicated that the cell growth and concentrations were not proportional to light intensity. During the 10 d culture period, both strains exhibited rapid growth initially, and then showed stagnation from days 4 and 6, respectively.

3.3. Effects of different light ratios (mixed red and blue LED) on the growth of H. Lacustris

Illumination with a red:blue light ratio of 2:1 (Table 3; Exp 4) was the most effective for the growth UTEX 2505, yielding a maximum cell concentration of 649,000 cells mL⁻¹ as shown in Fig. 7. The next most effective light ratios for the growth of UTEX 2505 were red:blue ratios of 3 : 1 and 1 : 1, which yielded 532,600 and 528,800 587,400 cells mL⁻¹, respectively (Fig. 7). The cell concentration under the red:blue ratio of 2 : 1 was approximately 21.8% and 22.7% higher than that under red:blue ratios of 3 : 1 and 1 : 1, respectively.

Figure 7. Concentration of Haematococcus lacustris cells under different ratios of mixed red and blue LEDs. (a) UTEX2505, (b) UTEX 16.

A red:blue LED ratio of 1 : 1 was the most effective for the growth of UTEX 16 (Fig. 7), yielding a maximum cell concentration of 370,800 cells mL⁻¹. The red:blue ratios of 2:1 and 1:2 yielded 352,200 and 350,400 cells mL⁻¹, respectively (Fig. 7). The cell concentration under the red:blue LED ratio of 1:1 was approximately 5.2% and 5.8% higher than that under the red:blue ratios of 2:1 and 1:2, respectively.

The growth rate of photosynthetic microorganisms depends on the intensity of light. In the present study, the effects of monochromatic light and mixed red and blue wavelength illumination at different intensities and ratios on H. lacustris biology were investigated. Monochromatic light experiments revealed changes in the growth rates corresponding to the light source. UTEX 2505 and UTEX 16 cells did not grow at 0 and 10 μmol m⁻² s⁻¹ light intensities under green LED and fluorescent light, and 0, 10, and 20 μmol m⁻² s⁻¹ light intensities under red LED. Furthermore, under 0 μmol m⁻² s⁻¹ blue LED light, the algal strains exhibited a decreasing trend of initial cell concentration with incubation. Additionally, in growth experiments, the highest cell concentrations were achieved under blue LED, followed by green LED and fluorescent lamp; the lowest cell concentrations were obtained under red LED. Previous studies [13,17] have reported that blue LED induces cell maturation and red LED induces cell division; however, in the present study, the cell concentrations were the lowest under red LED; this discrepancy in the results warrants further investigation.

The growth rate of H. lacustris was found to vary depending on the light intensity of different sources. Both UTEX 2505 and UTEX 16 strains did not grow under light sources of 0 and 10 μmol m⁻² s⁻¹ intensities; moreover, the cell concentrations showed a decreasing tendency with incubation time. In the culture experiments of UTEX 2505 and UTEX 16, when the light intensity was varied in the range of 0–300 μmol m⁻² s⁻¹, the highest cell concentrations were achieved at 100 μmol m⁻² s⁻¹ and lower cell concentrations were obtained at higher intensities of 200 and 300 μmol m⁻² s⁻¹. These results suggest that the growth of H. lacustris is not proportional to the intensity of light and that if the intensity of light is less than or exceeds a certain limit, it hinders cell growth.

In the present study, red and blue LED mixtures were conducive to the growth of H. lacustris. The red:blue LED ratio for obtaining the maximum cell concentration differed for UTEX 2505 and UTEX 16 strains. The highest growth of UTEX 2505 was achieved at a red:blue LED ratio of 2:1, whereas the highest growth of UTEX 16 was observed at a red:blue LED ratio of 1:1. The differences in the growth response of the two strains revealed in this study are likely attributable to differences in the characteristics of these strains. Further investigation is required to determine the specific growth-differentiating characteristics of the two strains.

Haematococcus lacustris is a representative microalgal species and a natural source of astaxanthin, which has anti-aging, anti-inflammatory, antioxidant, and immune-enhancing effects and is a common ingredient in health supplement foods. Several studies have been conducted to increase biomass productivity. Various environmental factors such as salinity, light, and nutrients reportedly affect astaxanthin biosynthesis. However, LED applications specifically in microalgal biotechnology remain limited. Therefore, in this study, we investigated the effect of various monochromatic light types, light intensities, and different ratios of mixed light on the growth of two strains of H. lacustris, UTEX 2505 and UTEX 16. The findings of this study provide basic data for investigating the complex and condition-dependent photosynthetic reactions of H. lacustris by optimizing the light source and intensity to improve biomass and astaxanthin production. This study revealed differences in the optimal growth conditions and growth responses of the two strains, highlighting the need to elucidate the relationship between the light ratio and growth rate of H. lacustris.

This research was supported by Korea Institute of Marine Science & Technology Promotion(KIMST) funded by the Ministry of Oceans and Fisheries, Korea(No. 20210505) and the Technology development Program of MSS [RS-2022-00167208].