A Study on the Effect of Various Media and the Supplementation of Organic Compounds on the Enhanced Production of Astaxanthin from Haematococcus lacustris (Girod—Chantrans) Rostafinski (Chlorophyta)

Natural astaxanthin is in high demand due to its multiple health benefits. The microalga Haematococcus lacustris has been used for the commercial production of astaxanthin. In this study, we investigated the effects of six different media with and without a nitrogen source and supplementation with nine organic compounds on the growth and astaxanthin accumulation of H. lacustris. The highest astaxanthin contents were observed in cultures of H. lacustris in Jaworski’s medium (JM), with a level of 9.099 mg/L in JM with a nitrogen source supplemented with leucine (0.65 g/L) and of 20.484 mg/L in JM without a nitrogen source supplemented with sodium glutamate (0.325 g/L). Six of the nine organic compounds examined (leucine, lysine, alanine, sodium glutamate, glutamine, and cellulose) enhanced the production of astaxanthin in H. lacustris, while malic acid, benzoic acid, and maltose showed no beneficial effects.


Introduction
Astaxanthin (3,3 ′ -dihydroxy-β-carotene-4,4 ′ -dione) is a red-orange pigment belonging to the carotenoid family with commercial applications in cosmetics, food supplements, livestock feedstuffs, and pharmaceuticals due to its remarkable biochemical characteristics, physiological effects, and physical properties [1,2].Although astaxanthin is naturally produced by a range of fungal and bacterial species, Haematococcus lacustris (Girod-Chantrans) Rostafinski (Chlorophyta) is considered the chief producer of astaxanthin among microalgae with astaxanthin contents up to 5% of cell dry weight [3].The large-scale production of astaxanthin from yeasts, fungi, bacteria, shrimp, fish, and so forth is not feasible because of their lower astaxanthin contents (less than 1% of dry weight) [4].Among microalgae, edible astaxanthin is mainly produced by Haematococcus lacustris, Halochlorella rubescens, Rhexinema sarcinoideum, Chromochloris zofingiensis, and Ettlia carotinosa [5].Haematococcus lacustris is an important microalga in terms of its astaxanthin production, biomass, and lipid production, which can be utilized for commercial purposes.Astaxanthin, a tetraterpene with linked isoprene units, consists of a linear polyene chain and two terminal β-ionone rings [1,[6][7][8][9].The presence of 13 conjugated double-bond systems causes it to have a pink and red color [1,6].The global astaxanthin market value was estimated to be USD 1 billion in 2019, which is anticipated to reach USD 3398.8 million by 2027 [8].Natural astaxanthin has properties that aid growth and enhance immunity as well as antioxidant, antiobesity, anticancer, antidiabetic, and antiinflammatory functions and protective effects against gastric ulcers and a number of ocular diseases.Due to these properties, it is used as a major bioactive component in beauty products, antiaging serums, and sunblock creams and is also used as an oral dietary supplement [6].H. lacustris cells undergo a number of ultrastructural changes throughout their life cycle depending on exposure to various stress conditions.Typically, the cells are spherical-ovoid in shape, with a diameter of approximately 30 µm.Initially, they are green palmelloid or biflagellate, freely swimming with a single pyrenoid-containing chloroplast.Later, the cells lose their flagella, round up into a nonmotile palmella, and turn into thick-walled aplanospores.The green-stage vegetative cells can reproduce asexually and give rise to 2 to 32 daughter cells.The accumulation of astaxanthin starts during the intermediate stage between days 7 and 10 when H. lacustris cells turn greenish-orange.There is an increase in the cell size and the loss of flagella due to stress conditions.The "red-astaxanthin formation" (red stage) appears between 11 and 14 days, where astaxanthin accumulation continues, with the cells forming cysts at the aplanospore stage [10].Then, a thick algeenan-containing cell wall is formed, which protects the aplanospore cells from acetolysis via high light exposure or nutrient deprivation.Finally, astaxanthin accumulates in droplets in the perinuclear cytoplasm, and the cells appear bright red in the mature aplanospore stage [10].
Some organic carbon sources, such as glucose and acetate, have been used for the supplementation of media in the commercial production of astaxanthin from H. lacustris (Girod-Chantrans) Rostafinski (Chlorophyta) [11].Supplementation with sodium acetate as an organic carbon source increases H. lacustris growth and astaxanthin accumulation.The addition of organic carbon sources to phototrophic growth cultures influences photosynthesis and carbon metabolism in H. lacustris [12].In a recent study, we evaluated the effects of four organic carbon sources: ribose, glycerol, sodium acetate, and sodium gluconate [5].Astaxanthin accumulation is enhanced in H. lacustris by supplementation with glycerol [13], sodium fumarate [14], trisodium citrate [15], and oxaloacetate [16] under various conditions.Sodium glutamate, or monosodium glutamate (MSG), is a widely used food enhancer that was originally extracted from beet sugar but has also been obtained by the microbial fermentation of potato starch, cassava, wheat, and so forth [17].Microalgae produce high levels of carotenoids when mariculture wastewater is modified with sodium glutamate residue [18].Adding sodium glutamate to a medium is beneficial for the growth of Spirulina maxima (now classified as Limnospira maxima) [19,20].Malic acid is a dicarboxylic acid with four carbons that has biological phytohormone effects and promotes cold resistance and the growth of seedlings [21].Leucine is an important nutrient in plants that plays a role in signalling for lipid decomposition, protein metabolism, biomass accumulation, and other biological reactions [22].Glutamine affects gene expression in plants, and at low concentrations, it has been shown to improve the growth of rice seedlings [23].Benzoic acid inhibits plant growth and plays a major role in crop autotoxicity [24].Cellulose is a simple polymer and the most abundant carbohydrate produced by plants.It is highly resistant to enzymatic hydrolysis and forms insoluble, crystalline microfibrils.A complete set of enzymes with the ability to effectively degrade cellulose is present only in a few microorganisms, such as eubacteria, fungi, anaerobic cellulose-degrading protozoa, avocado fruit, and slime mold (Dictyostelium discoideum) [25] Moreover, Chlamydomonas reinhardtii, a phototrophic unicellular green microalga belonging to the order Chlamydomonadales and the class Chlorophyceae, has the ability to utilize cellulose for its growth in the absence of other carbon sources and digest exogenous cellulose when grown under CO 2 -limiting conditions in the light [26].Alanine improves lipid production and biomass accumulation in microalgae [27].Lysine plays a number of different metabolic roles in plants [28].The effects of maltose have been studied in Auxenochlorella pyrenoidosa (formerly Chlorella pyrenoidosa), and it has been shown to increase the growth of Botryococcus braunii and the cell density of A. pyrenoidosa [29,30].
In this study, we used six different media that have been utilized for the growth of microalgae in recent studies.We compared and evaluated the effects of these media on the growth and astaxanthin accumulation of Haematococcus lacustris (Girod-Chantrans) Rostafinski (Chlorophyta) under laboratory conditions.As our preliminary experiments showed that H. lacustris produced more astaxanthin in Jaworski's medium (JM) under nitrogen-depleted conditions compared to other media, this was used as the basal medium.The other consideration of using JM was its economical profitability.Next, we examined the effects of the supplementation of JM with different readily available organic compounds on growth and astaxanthin accumulation in H. lacustris.

Microalgae and Culture Conditions
H. lacustris (LIMS-PS-1354) was obtained from the Library of Marine Samples at The Korea Institute of Ocean Science and Technology (KIOST), Geoje, Republic of Korea, and cultured and maintained at 21 ± 1 • C under a 12/12 h light-dark cycle and a photon flux density of 37 µmol m −2 s −1 in (JM).
For the preparation of media without nitrogen sources, Walne/Conway, OHM, modified Chu-13, and JM were prepared without KNO 3 .Similarly, BG-11, BBM, and JM were prepared without NaNO 3 .The constituents of the six media are listed in Table 1.The pH of the modified CHU 13, BG-11, JM, BBM, Walne/Conway, and OHM were adjusted to 6.7, 7.5, 7.0, 6.8, 7.5, and 7.0, respectively.The experiments were carried out in three replicates in 40 mL vials containing 30 mL of the medium and 10 mL of the seed culture.A 4-day-old culture was used as the seed culture for the initial inoculum.The media experiment was replicated five times, and the experiments with the supplementation of organic carbon compounds were replicated three times.The experiment was carried out over a period of 14 days.The effects of supplementation with the following nine organic compounds were examined: sodium glutamate, malic acid, leucine, glutamine, benzoic acid, cellulose, lysine, alanine, and maltose.All nine of these organic compounds are carbon sources, while five (alanine, leucine, lysine, glutamine, and sodium glutamate) are also nitrogen sources.These compounds were added to the media at concentrations of 0 g/L (control), 0.325 g/L, 0.65 g/L, 1.3 g/L, and 2.6 g/L.

Experimental Design
H. lacustris was cultured under a photon flux density of 40 µmol m -2 s -1 .The fluorescent light intensity was measured using a light meter (Fluke-941; Fluke Corp., Everett, WA, USA).The experiments were performed at 16

Analysis of the Cell Number and Astaxanthin Accumulation
A haemocytometer with an improved Neubauer chamber (Marienfeld Superior, Lauda-Konigshofen, Germany) and a light microscope with a 20× objective lens (BX53; Olympus, Tokyo, Japan) were used to estimate the cell number.The analysis of astaxanthin was performed according to the methods described in previous studies [5,6].

Statistical Analysis
The cell number and pigment data are represented as the mean ± standard deviation.Statistical analyses were performed using one-way ANOVA followed by Tukey's multiple comparison test using SPSS (version 22; IBM Corp., Armonk, NY, USA).In all analyses, p < 0.001 was taken to indicate statistical significance.

Effect of Media on the Growth of H. lacustris
During the cultivation in BBM with a nitrogen source (+N), the H. lacustris cell cultures were green until day 9 but turned orange on day 12.However, the cultures remained green until day 12 when cultivated in BBM without a nitrogen source (-N) (Figure 1).During cultivation in JM (+N) and JM (−N), the cultures were green until day 6 but turned orange on day 9 and pink on day 12.During cultivation in BG-11 (+N), they were green until day 12.In BG-11 (−N), however, they remained green until day 9 but turned yellowish orange on day 12.During cultivation in Walne/Conway (+N), the cultures were green until day 9 and turned pink on day 12.In Walne/Conway (−N), they remained green until day 6, turned orange on day 9, and turned pink on day 12.In OHM (+N), they were green until day 12.In OHM (−N), they remained green until day 9 but turned yellowish-orange on day 12.In Chu-13 (+N), they were green until day 9 but turned yellowish-orange on day 12.In Chu-13 (−N), they remained green until day 9 and turned orange on day 12.

Statistical Analysis
The cell number and pigment data are represented as the mean ± standard deviation.Statistical analyses were performed using one-way ANOVA followed by Tukey's multiple comparison test using SPSS (version 22; IBM Corp., Armonk, NY, USA).In all analyses, p < 0.001 was taken to indicate statistical significance.

Effect of Media on the Growth of H. lacustris
During the cultivation in BBM with a nitrogen source (+N), the H. lacustris cell cultures were green until day 9 but turned orange on day 12.However, the cultures remained green until day 12 when cultivated in BBM without a nitrogen source (-N) (Figure 1).During cultivation in JM (+N) and JM (−N), the cultures were green until day 6 but turned orange on day 9 and pink on day 12.During cultivation in BG-11 (+N), they were green until day 12.In BG-11 (−N), however, they remained green until day 9 but turned yellowish orange on day 12.During cultivation in Walne/Conway (+N), the cultures were green until day 9 and turned pink on day 12.In Walne/Conway (−N), they remained green until day 6, turned orange on day 9, and turned pink on day 12.In OHM (+N), they were green until day 12.In OHM (−N), they remained green until day 9 but turned yellowish-orange on day 12.In Chu-13 (+N), they were green until day 9 but turned yellowish-orange on day 12.In Chu-13 (−N), they remained green until day 9 and turned orange on day 12.

Effect of Media on Astaxanthin Levels in H. lacustris
The astaxanthin levels were the highest for JM (−N) and the lowest for OHM (+N).The astaxanthin production by H. lacustris grown in JM (−N) was approximately 15% higher than that in Conway/Walne (−N), 22% higher than that in Chu-13 (−N), 50% higher

Effect of Media on Astaxanthin Levels in H. lacustris
The astaxanthin levels were the highest for JM (−N) and the lowest for OHM (+N).The astaxanthin production by H. lacustris grown in JM (−N) was approximately 15% higher than that in Conway/Walne (−N), 22% higher than that in Chu-13 (−N), 50% higher than that in BBM (−N), 183% higher than that in BG-11 (−N), and 158% higher than that in OHM (−N) (Figure 3).than that in BBM (−N), 183% higher than that in BG-11 (−N), and 158% higher than that in OHM (−N) (Figure 3).

Effects of Organic Compounds on H. lacustris Cell Numbers and Astaxanthin Levels
H. lacustris was grown in JM (+N) and JM (−N) supplemented with various organic compounds, and the cell numbers are shown in Tables 2-19.When grown in JM (+N), the cell numbers increased significantly under all conditions, being much higher than those in the controls on all days.For example, on day 6, the cell numbers were increased by 85.92-fold in cultures with leucine (0.325 g/L), 48.27-fold in cultures with leucine (0.65 g/L), 37.82-fold in cultures with sodium glutamate (0.325 g/L), 174.38-fold in cultures with maltose (0.325 g/L), 42-fold in cultures with maltose (0.325 g/L), 75-fold in cultures with maltose (0.65 g/L), 58.77-fold in cultures with maltose (1.3 g/L), and 37.82-fold in cultures with maltose (2.6 g/L).The numbers were similarly high for the other days.
There were no differences in the cell number in cultures grown in JM (−N) supplemented with various organic compounds compared to that of JM (+N), with the exception of a 51.35
In this experiment, astaxanthin levels were higher after growth in JM (+N) than those after growth in JM (−N).The levels grown with lysine (1.3 g/L), sodium glutamate (0.325 g/L), cellulose (0.325 g/L), cellulose (0.65 g/L), and cellulose (2.6 g/L) were higher than the control level of 11.637 mg/L, and the highest astaxanthin content of 20.484 mg/L was observed when grown in the presence of sodium glutamate at 0.325 g/L.

Discussion
Cultivation under conditions of nitrogen starvation is considered necessary to produce astaxanthin from H. lacustris.As JM showed the highest concentration of astaxanthin (5.87 mg/mL) under nitrogen-depleted conditions, we found that JM (−N) was the optimal medium for astaxanthin production by H. lacustris.Moreover, another reason for selecting JM was its cost-effectiveness on a larger scale.As we can see, the concentrations of the chemical ingredients required for JM are lower.However, when we compared the cell numbers and astaxanthin production of H. lacustris during the organic compounds supplementation experiment with the media experiment, the results showed that the cell numbers and astaxanthin production of H. lacustris grown under JM (+N) were higher than those under JM (−N).This could be dependent on the initial cell number of H. lacustris.The initial cell numbers of H. lacustris grown under JM (+N) and JM (−N) for the media experiment were higher by 15.45-fold and 10.05-fold, respectively, than for the organic compounds supplementation experiment.Therefore, there is still a further requirement for additional experiments to optimize the initial cell numbers for the commercial production of astaxanthin from H. lacustris.Five of the nine organic carbon sources tested as supplements-alanine, leucine, lysine, glutamine, and sodium glutamate-contain nitrogen.Nitrogen has been shown to play an important role in both cell growth and astaxanthin accumulation by H. lacustris [39].A previous study examined the effects of four organic carbon sources on astaxanthin production by H. lacustris, i.e., glycerol, sodium acetate, ribose, and sodium gluconate [5].Nitrogen is a vital nutrient in the growth medium of H. lacustris.Its depletion induces stress responses, as a result of which the green motile cells stop dividing and turn into hematocysts ready for the accumulation of astaxanthin with the increased activity of the Tricarboxylic acid (TCA) cycle [40,41].The range of optimal light intensity for microalgae is between 26 and 400 µmol photons m −2 s −1 [42].Nitrogen plays an essential role in the cultivation of microalgae since it is a critical component of biological macromolecules such as DNA, protein, and chlorophyll.Nitrogen deficiency can enhance lipid accumulation and lower the biomass yield [43]. Astaxanthin accumulated in H. lacustris under growth-limiting conditions, such as high salinity, high light intensity, and nutrient deficiency.Nitrogen is also an essential nutrient responsible for enzymatic activities and the cell growth of H. lacustris.Its deficiency inhibits the biosynthesis of chlorophyll and thus impairs photosynthetic function [44].Moreover, we also evaluated the effects of a low light intensity and lower temperature coupled with different media and supplementation with organic compounds on the growth and astaxanthin accumulation of H. lacustris.Cultures were illuminated with cool-white fluorescent lamps with an intensity of 40 µmol m −2 s −1 [12], and the experiments were performed at the relatively low temperature of 16 When H. lacustris was grown in JM (+N) media supplemented with various organic compounds, the greatest increment in cell number (174.38-fold) was observed on day 6 in the culture with maltose (0.325 g/L).On day 9, the greatest increment in the cell number (129.76-fold) was again seen with maltose (0.325 g/L), while on day 12, the greatest increment in the cell number (115.65-fold) was seen with leucine (1.3 g/L).Similarly, when grown in JM (−N) supplemented with various organic compounds, the greatest increment in the cell number (51.35-fold) was seen with sodium glutamate (0.325 g/L) on day 6.The greatest increment in the cell number on day 9 (59.35-fold) was again seen with sodium glutamate (0.325 g/L), while on day 12, the greatest (126.15-fold) was seen with leucine (1.3 g/L).The results showed improvements in astaxanthin production by H. lacustris in JM (−N) supplemented with alanine (0.325 g/L, 0.65 g/L, 1.3 g/L), lysine (2.6 g/L), leucine (0.325 g/L, 0.65 g/L, 1.3 g/L, 2.6 g/L), glutamine (0.325 g/L, 0.65 g/L, 1.3 g/L), and sodium glutamate (0.325 g/L, 0.65 g/L, 1.3 g/L) compared to a previous study that supplemented the culture medium with ribose, sodium acetate, sodium gluconate, and glycerol [5].Similarly, when cultivated in JM (+N) supplemented with various organic compounds, H. lacustris showed increases in astaxanthin contents of 1.17-fold in a medium supplemented with lysine (1.3 g/L), 1.75-fold in a medium supplemented with sodium glutamate (0.325 g/L), 1.52-fold in a medium supplemented with cellulose (0.325 g/L), 1.04-fold in a medium supplemented with cellulose (0.65 g/L), and 1.08-fold in a medium supplemented with cellulose (2.6 g/L) compared to the control.These observations suggest that astaxanthin production can be improved even without the removal of nitrogen sources from JM.

Conclusions
Culture in JM (−N) supplemented with leucine (0.65 g/L) and sodium glutamate (0.325 g/L) significantly enhanced the accumulation of astaxanthin in H. lacustris.Astaxanthin production was increased in JM (+N) supplemented with lysine (1.3 g/L), sodium glutamate (0.325 g/L), cellulose (0.325 g/L), cellulose (0.65 g/L), and cellulose (2.6 g/L).Similarly, when cultured in JM (−N), its production was increased by the supplementation of the medium with alanine (0.325 g/L, 0.65 g/L, 1.3 g/L), lysine (2.6 g/L), leucine (0.325 g/L, 0.65 g/L, 1.3 g/L, 2.6 g/L), glutamine (0.325 g/L, 0.65 g/L, 1.3 g/L), and sodium glutamate (0.325 g/L, 0.65 g/L, 1.3 g/L).Overall, the supplementation of four amino acids (glutamine, alanine, lysine, and leucine) along with sodium glutamate and cellulose enhanced the astaxanthin accumulation in H. lacustris.Glutamate feeding enhanced the yield of astaxanthin in Xanthophyllomyces dendrorhous.Apart from serving as a nitrogen source in a cell, it also participates in the TCA cycle [45].There is a great deal of interest in astaxanthin production from microalgae due to the health benefits of natural astaxanthin.Further studies are required on the solubility of cellulose in JM and to determine the mechanisms underlying the effects of supplementation with lysine, leucine, glutamine, alanine, and cellulose on the growth and astaxanthin accumulation of H. lacustris at the molecular level.

Figure 2 .
Figure 2. (A) Comparison of the cell number of H. lacustris under the influence of different media with nitrogen source (0D-Day 0, 3D-Day 3, 6D-Day 6, 9D-Day 9, 12D-Day 12).(B) Comparison of the cell number of H. lacustris under the influence of different media without nitrogen source (1D-Day 1, 3D-Day 3, 6D-Day 6, 9D-Day 9, 12D-Day 12).Mean values that do not share the same letter are significantly different at p < 0.001.

Figure 2 .
Figure 2. (A) Comparison of the cell number of H. lacustris under the influence of different media with nitrogen source (0D-Day 0, 3D-Day 3, 6D-Day 6, 9D-Day 9, 12D-Day 12).(B) Comparison of the cell number of H. lacustris under the influence of different media without nitrogen source (1D-Day 1, 3D-Day 3, 6D-Day 6, 9D-Day 9, 12D-Day 12).Mean values that do not share the same letter are significantly different at p < 0.001.

Figure 3 .
Figure 3. Astaxanthin accumulation by H. lacustris grown in different media in 14 days (A) with a nitrogen source and (B) without a nitrogen source.Mean values that do not share the same letter are significantly different at p < 0.001.

Figure 3 .
Figure 3. Astaxanthin accumulation by H. lacustris grown in different media in 14 days (A) with a nitrogen source and (B) without a nitrogen source.Mean values that do not share the same letter are significantly different at p < 0.001.

Table 1 .
Chemical composition of six different media used in this study. 2

Table 2 .
Cell number of H. lacustris grown in JM (+N) media (Day 1, Day 3, Day 6, Day 9, Day 12) and astaxanthin contents of H. lacustris on Day 14 grown with the supplementation of benzoic acid.Data are presented as the mean ± standard deviation.

Table 3 .
Cell number of H. lacustris grown in JM (+N) media (Day 1, Day 3, Day 6, Day 9, Day 12) and astaxanthin contents of H. lacustris on Day 14 grown with the supplementation of cellulose.Data are presented as the mean ± standard deviation.Lowercase letters indicate significant differences (p < 0.001). H.

Table 4 .
Cell number of H. lacustris grown in JM (+N) media (Day 1, Day 3, Day 6, Day 9, Day 12) and astaxanthin contents of H. lacustris on Day 14 grown with the supplementation of glutamine.Data are presented as the mean ± standard deviation.Lowercase letters indicate significant differences (p < 0.001).H.

Table 5 .
Cell number of H. lacustris grown in JM (+N) media (Day 1, Day 3, Day 6, Day 9, Day 12) and astaxanthin contents of H. lacustris on Day 14 grown with the supplementation of alanine.Data are presented as the mean ± standard deviation.Lowercase letters indicate significant differences (p < 0.001).

Table 6 .
Cell number of H. lacustris grown in JM (+N) media (Day 1, Day 3, Day 6, Day 9, Day 12) and astaxanthin contents of H. lacustris on Day 14 grown with the supplementation of leucine.Data are presented as the mean ± standard deviation.Lowercase letters indicate significant differences (p < 0.001).

Table 7 .
Cell number of H. lacustris grown in JM (+N) media (Day 1, Day 3, Day 6, Day 9, Day 12) and astaxanthin contents of H. lacustris on Day 14 grown with the supplementation of lysine.Data are presented as the mean ± standard deviation.Lowercase letters indicate significant differences (p < 0.001).H.

Table 8 .
Cell number of H. lacustris grown in JM (+N) media (Day 1, Day 3, Day 6, Day 9, Day 12) and astaxanthin contents of H. lacustris on Day 14 grown with the supplementation of malic acid.

Table 9 .
Cell number of H. lacustris grown in JM (+N) media (Day 1, Day 3, Day 6, Day 9, Day 12) and astaxanthin contents of H. lacustris on Day 14 grown with the supplementation of maltose.Data are presented as the mean ± standard deviation.Lowercase letters indicate significant differences (p < 0.001).

Table 10 .
Cell number of H. lacustris grown in JM (+N) media (Day 1, Day 3, Day 6, Day 9, Day 12) and astaxanthin contents of H. lacustris on Day 14 grown with the supplementation of sodium glutamate.Data are presented as the mean ± standard deviation.Lowercase letters indicate significant differences (p < 0.001). H.

Table 11 .
Cell number of H. lacustris grown in JM (−N) media (Day 1, Day 3, Day 6, Day 9, Day 12) and astaxanthin contents of H. lacustris on Day 14 grown with the supplementation of benzoic acid.Data are presented as the mean ± standard deviation.H.

Table 12 .
Cell number of H. lacustris grown in JM (−N) media (Day 1, Day 3, Day 6, Day 9, Day 12) and astaxanthin contents of H. lacustris on Day 14 grown with the supplementation of cellulose.Data are presented as the mean ± standard deviation.Lowercase letters indicate significant differences (p < 0.001).

Table 13 .
Cell number of H. lacustris grown in JM (−N) media (Day 1, Day 3, Day 6, Day 9, Day 12) and astaxanthin contents of H. lacustris on Day 14 grown with the supplementation of glutamine.Data are presented as the mean ± standard deviation.Lowercase letters indicate significant differences (p < 0.001).

Table 14 .
Cell number of H. lacustris grown in JM (−N) media (Day 1, Day 3, Day 6, Day 9, Day 12) and astaxanthin contents of H. lacustris on Day 14 grown with the supplementation of alanine.Data are presented as the mean ± standard deviation.Lowercase letters indicate significant differences (p < 0.001).

Table 15 .
Cell number of H. lacustris grown in JM (−N) media (Day 1, Day 3, Day 6, Day 9, Day 12) and astaxanthin contents of H. lacustris on Day 14 grown with the supplementation of leucine.Data are presented as the mean ± standard deviation.Lowercase letters indicate significant differences (p < 0.001).

Table 16 .
Cell number of H. lacustris grown in JM (−N) media (Day 1, Day 3, Day 6, Day 9, Day 12) and astaxanthin contents of H. lacustris on Day 14 grown with the supplementation of lysine.Data are presented as the mean ± standard deviation.Lowercase letters indicate significant differences (p < 0.001).

Table 17 .
Cell number of H. lacustris grown in JM (−N) media (Day 1, Day 3, Day 6, Day 9, Day 12) and astaxanthin contents of H. lacustris on Day 14 grown with the supplementation of malic acid.Data are presented as the mean ± standard deviation.Lowercase letters indicate significant differences (p < 0.001).

Table 18 .
Cell number of H. lacustris grown in JM (−N) media (Day 1, Day 3, Day 6, Day 9, Day 12) and astaxanthin contents of H. lacustris on Day 14 grown with the supplementation of maltose.Data are presented as the mean ± standard deviation.Lowercase letters indicate significant differences (p < 0.001). H.

Table 19 .
Cell number of H. lacustris grown in JM (−N) media (Day 1, Day 3, Day 6, Day 9, Day 12) and astaxanthin contents of H. lacustris on Day 14 grown with the supplementation of sodium glutamate.Data are presented as the mean ± standard deviation.Lowercase letters indicate significant differences (p < 0.001).