Effect of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid on different growth stages of Haematococcus pluvialis

Accepted Manuscript Effect of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid on different growth stages of Haematococcus pluvialis Thi-Thao Vo, Changsu Lee, Sang-Il Han, Jee Young Kim, Sok Kim, Yoon-E Choi PII: DOI: Reference:

S0960-8524(16)31174-9 http://dx.doi.org/10.1016/j.biortech.2016.08.046 BITE 16949

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Bioresource Technology

Received Date: Revised Date: Accepted Date:

21 June 2016 9 August 2016 10 August 2016

Please cite this article as: Vo, T-T., Lee, C., Han, S-I., Kim, J.Y., Kim, S., Choi, Y-E., Effect of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid on different growth stages of Haematococcus pluvialis, Bioresource Technology (2016), doi: http://dx.doi.org/10.1016/j.biortech.2016.08.046

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Effect of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid on different growth stages of Haematococcus pluvialis Thi-Thao Vo1, Changsu Lee2, Sang-Il Han3, Jee Young Kim3, Sok Kim3, and Yoon-E Choi3⃰. Department of Bioactive Material Sciences, Chonbuk National University1 Department of Bioprocess Engineering, Chonbuk National University2 Division of Environmental Science & Ecological Engineering, Korea University3 (1) and (2) contributed equally to this study *Corresponding author. Tel: +82 2 3290 3042, Fax: +82 2 3290 3040, E-mail: [email protected]

Abstract

In this study, we explored the effects of ACC on other stages of H. pluvialis. Interestingly, even though ACC displayed a dose-dependent effect on astaxanthin production, it is evident that astaxanthin production could be facilitated whenever the cells were treated at the early red stage. The transcriptional levels of BKT, CHY, SOD, and CAT genes supported enhanced astaxanthin biosynthesis upon ACC treatment at the early red stage. The combinatorial synergistic effect of ACC and light intensity was also confirmed. Finally, two-step application of ACC at the vegetative phase to increase biomass production and at the early-red stage to promote astaxanthin biosynthesis was proposed to maximize the efficiency of ACC treatment.

Keywords: Haematococcus pluvialis, astaxanthin, 1-aminocyclopropane-1-carboxylic acid, two-step applications

I.

Introduction

The unicellular green microalga Haematococcus pluvialis, is a well-known source of astaxanthin (3,3′-dihydroxy-β,β-carotene-4,4′-dione), a ketocarotenoid that is used as a food additive for the pink-red pigmentation of salmonids, shrimp, lobsters, and crayfish (Lorenz & Cysewski, 2000). Astaxanthin is also demonstrated to possess antioxidant properties, such as ROS scavenging and free radical neutralization, which are ten times higher than other carotenoids. Due to its significant effect, astaxanthin has been used in the clinic to fight against oral, skin, colon, and liver cancers, cardiovascular diseases, and degenerative eye diseases (Guerin et al., 2003; Lorenz & Cysewski, 2000).

The life cycle of H. pluvialis consists of four stages: vegetative (green), encystment (immature cyst/brown), maturation (cyst/red), and germination followed by the vegetative stage again (Kobayashi et al., 1997). Vegetative cells are spherical, ellipsoidal, or pearshaped with two flagella and cup-shaped chloroplasts. At this stage, they grow rapidly and each cell divides into 2-8 daughter cells; the cells contain high levels of chlorophyll and protein. When the environmental conditions become unfavorable, the cells lose their flagella and drastically increase their volume to become spherical, immotile, immature cyst cells, where the carotenoid content is increased. The maturation of cyst cells includes the thickening of cell walls, enhanced biosynthesis of carotenoids and hastened protein degradation. When the red cells are subjected to favorable conditions, intracellular daughter cells were formed and then released, thus initiating a new cycle (Danxiang et al., 2013; Kobayashi et al., 1997).

In H. pluvialis, 1-aminocyclopropane-1-carboxylic acid (ACC) was indicated to be the immediate precursor of ethylene in the biosynthetic pathway (Maillard et al., 1993).

Ethylene is often referred to as an aging hormone in plants. Various studies have demonstrated the inducing role of ethylene in fruit ripening (Barry & Giovannoni, 2007; Yang & Hoffman, 1984) as well as in carotenoid accumulation (Rodrigo & Zacarias, 2007; Stewart & Wheaton, 1972). Meanwhile, few studies have been conducted regarding to exogenous treatment of ethylene or ACC on microalgae. For examples, Gao and Meng (2007) applied exogenous ethylene to vegetative cells of H. pluvialis under stress conditions. The study by Gao and Meng is the only study performed for astaxanthin production using ethylene or ACC. However, the study (Gao and Meng, 2007) simply investigated a possible positive effect of ethylene on astaxanthin biosynthesis, without considering different cell growth stages of H. pluvialis. In addition, ethephon (an ethylene releaser) is also utilized as a replacement of the ethylene treatment, when ethylene is not available. The presence of ethephon in Chlorella vulgaris culture resulted in significantly higher levels of α-tocopherol, γ-aminobutyric acid, asparagine, proline, and saturated fatty acids, but lower levels of glycine, citrate, galactose and unsaturated fatty acids (Kim et al., 2016). Other than these studies, no information is available so far on the ethylene or ACC application of astaxanthin production.

Our previous study regarding ACC treatment on the vegetative cells of H. pluvialis revealed a growth promoting effect, thereby enhancing astaxanthin production (manuscript under preparation). However, since gradual accumulation of astaxanthin occurs during transition from the brown stage to the red cyst stage, we hypothesized that ACC might have additional facilitating or different effects depending on the cell stage of H. pluvialis. Thus, the detailed objectives of this study were: (1) to investigate the impact of ACC on the brown and the red stages in terms of growth and astaxanthin production; (2) to explore the varied extent of astaxanthin production via ACC treatment depending on either the brown

or red stage; (3) to examine the possible combinatorial effect of ACC and different light intensities on astaxanthin production, since light is the most significant factor in astaxanthin biosynthesis; (4) in summary, we attempted to propose a novel strategy suggesting the appropriate supplementation of ACC at different growth stages to enhance astaxanthin productivity in H. pluvialis.

II. Materials and methods

2.1. Algal strains and culture conditions

The unicellular green alga, H. pluvialis (UTEX# 2505) was obtained from the culture collection of algae at the University of Texas, Austin, Texas, USA. The strains were regularly cultivated in a 250 mL round-bottomed flask containing 150 mL of sterile OHM medium (Fábregas et al., 2000), composed of 0.41 g KNO3, 0.03 g Na2HPO4, 0.246 g MgSO4.7H2O, 0.11 g CaCl2.2H2O, 2.62 mg Fe(III)-citrate.H2O, 0.011 mg CoCl2.6H2O, 0.12 mg CuSO4.5H2O,0.075 mg Cr2O3, 0.98 mg MnCl2.4H2O, 0.12 mg Na2MoO4.2H2O, 0.005 mg SeO2, 25 g biotin, 17.5 g thiamine, and 15 g B12, per liter of deionized distilled water. Cultivation was performed with 1.5 vvm aeration at 25°C. The inoculum size at the vegetative stages was adjusted to 10×104 cells/mL.

2.2. ACC treatment

To define the effect of ACC on H. pluvialis, cell growth, biomass, and the varying extent of astaxanthin production was analyzed upon ACC treatment. Selected concentrations of ACC (0.1, 1, and 5 mmol/L) were added to the algal cultures either at the early brown or at the early red stage. Samples without ACC supplementation served as controls. The experiments were carried out for more than three biological replicates. To perform the experiments, low light intensity (20 µmol m-2s-1) was chosen to rule out any complicating effect of light intensity to increase astaxanthin production using H. pluvialis.

Growth determination

Dry weight was measured by filtering an aliquot of the algal suspension through a predried and pre-weighed 0.45 µm cellulose nitrate membrane filter (Whatman, USA) and drying in an oven at 80oC for 10 h. The number of cells was determined by cell counting using an improved Neubauer hemocytometer.

Pigment measurement

The cells were harvested and homogenized in 90% acetone using a bead-beater. The extract was collected by centrifugation and astaxanthin content was determined by a spectrophotometer (OD474). An astaxanthin standard (Sigma-Aldrich, U.S.A.) was used to generate a standard curve. The level of astaxanthin production was calculated using the following equation:

Astaxanthin (mg/L) = 7.0881 * OD474 - 0.5904

Total chlorophyll was extracted by the same method and was determined according to the equations reported by Li et al. (2000):

Chl a (mg/L) = 12.21 * OD663 - 2.81 * OD646

Chl b (mg/L) = 20.13 * OD646 - 5.03 * OD663

Total chlorophyll (mg/L) = Chl a + Chl b

2.3. Transcript levels of BKT, CHY, CAT, and SOD genes

Total RNA was extracted from H. pluvialis cells using TRIzol reagent (Invitrogen) following the manufacturer’s protocol. Nucleic acid concentrations were measured spectrophotometrically at 260 nm. RNA integrity and purity were determined by the 260 nm/280 nm ratio and were separated by electrophoresis on 1% agarose gel. cDNA was synthesized with a high-capacity RNA-to-cDNA kit (Applied Biosystems). Total RNA (5 µg) and 2X RT enzyme mixture were mixed in a reaction tube and incubated at 37°C for 1 h, inactivated at 95°C for 5 min, and then quickly chilled on ice.

The expression of genes encoding ROS scavenging enzymes like SOD (superoxide dismutase) (NCBI Gene ID: AY878538) and CAT (NCBI Gene ID: EF043383) and genes related to astaxanthin biosynthesis including BKT (NCBI Gene ID: D45881) and CHY (NCBI Gene ID: AF162276), was measured by qRT-PCR using SYBR-Green® as the fluorescent dye. The amplification was performed by 40 cycles of 95°C for 20 s, 60°C for 20 s using StepOnePlus (Thermo Fisher Scientific). The gene-specific primers that were used are presented in Table 1. The expression of each gene was normalized to endogenous actin expression (NCBI Gene ID: DV203941).

2.4. Effect of ACC under different light intensities

The possible combinatorial effects of ACC treatment and light intensity were investigated. The early red cells were subjected to ACC treatment simultaneously with illumination at various light intensities. Briefly, the early red-stage cultures were supplemented with 1 mmol/L ACC and were placed under different light intensities, i.e. 20, 40, and 80 µmol m-2 s-1. The time course of changes in astaxanthin accumulation was thoroughly monitored after this treatment.

2.5. Two-step supplementation of ACC to H. pluvialis culture

In order to examine the putative serial applications of ACC depending on the growth stage of H. pluvialis, two different concentration of ACC (0.1 mmol/L and 1 mmol/L) were initially supplemented to H. pluvialis cultures. The inoculum was prepared with 1 × 105 cells/ml. The experiments were conducted in 250 mL flasks with 1.5 vvm aeration under a light intensity of 60 µmol m-2 s-1. After reaching the early cyst stage, further applications of ACC (either 0.1 mmol/L or 1 mmol/L) were performed and subsequent growth and astaxanthin production were monitored. As control, a culture without ACC treatment was also included following all the experimental conditions described above except those for ACC treatment.

III. Results and Discussion

3.1. ACC promoted biomass production in H. pluvialis

We first grew H. pluvialis and obtained a prolonged time scale of its growth curve, incorporating the cell number, biomass, and astaxanthin content together (Fig. 1A). The growth curve of H. pluvialis showed that the vegetative stage lasted for approximately 10 days and subsequently reached the maturation stage. Based on the H. pluvialis growth curve, we could determine the possible treatment time for ACC in order to test the putative effect of ACC on either the early brown stage or the early red stage. The early brown stage, which is at the end of the exponential phase, was marked when cells lost flagella and formed round shape with thick cell wall. At this stage, cells began turning to a brownish color, indicating the initial carotenoid production (Fig. 1B). The early red stage, which is at the mid-stationary phase, was recognized, when large red spots appeared in cells, rendering the cells culture reddish (Fig. 1C).

ACC treatment caused an increase in chlorophyll content and biomass, which were observed both across the early brown and the early red stage. Regardless of H. pluvialis stage, increase in chlorophyll as well as biomass production was proportional to the extent of ACC treatment, indicating the positive effect of ACC on H. pluvialis growth, as observed with the effect of ACC on the vegetative stage previously (Fig. 2A-D). Figure 2A and 2B show the striking increase in chlorophyll with 5 mmol/L ACC treatment and the highest amounts in the brown and red stages were 1.9-fold and 2-fold higher than those in the controls, respectively. Our data suggests that ACC caused a substantial increment in H. pluvialis growth regardless of growth stage. Even though ACC at a lower concentration of below 0.1 mmol/L had a negligible influence on growth, it is evident that ACC

supplementation at above a certain concentration could result in increased growth of H. pluvialis even at the brown and red cell stages. Our results suggest the interesting application of ACC for enhancing the growth of H. pluvialis further, even after the matured cyst stage.

Ethylene is known to exhibit either an inhibitory (Fiorani et al., 2002; Guzmán & Ecker, 1990; Stepanova & Alonso, 2005) or a stimulatory effect (Kende et al., 1998; Pierik et al., 2003; Smalle et al., 1997) on growth in many plants, depending on environmental conditions, developmental stage, organs, and species specificity. However, our data evidently suggests that ethylene, which is the final product of ACC, might be helpful to increase H. pluvialis growth, instead of similar growth inhibitory effects as in plants. In case of H. pluvialis, photosynthesis is still active without cell division during cyst formation, possibly resulting in the readiness to respond to exogenous ACC resulting in increased cell size and mass (Hagen et al., 2000; Qiu & Li, 2006). We speculated that this might explain the increased patterns of chlorophyll and biomass production observed in H. pluvialis. Further studies will be necessary to pinpoint the exact mechanisms involved in different cellular stages of H. pluvialis upon ACC or ethylene treatment.

3.2. Dose and cell-stage dependent effect of ACC on astaxanthin production

Our results revealed a different pattern in case of astaxanthin production, compared to observations with H. pluvialis growth. At the brown stage, only low ACC concentrations, 0.1 mmol/L and 1 mmol/L, could enhance the biosynthesis of ketocarotenoid, while 5 mmol/L slowed this process. On day 15, astaxanthin content was increased by 11% and

22% in 0.1 mmol/L and 1 mmol/L ACC-treated groups, respectively; however, in 5 mmol/L, the content was about 7% lower than that observed in control (Fig. 2E). Therefore, ACC treatment at the brown stage did not show a remarkable increase in astaxanthin production as was expected.

However, interestingly, when ACC was added to the red stage, all ACC concentrations could stimulate astaxanthin production significantly (Fig. 2F). The impact order was 1 mmol/L > 0.1 mmol/L > 5 mmol/L > control. Compared to the brown stage, ACC treatment at the red stage resulted in a distinguishable effect at 6 days. After 15 days, astaxanthin content was increased by 77%, 43%, and 35% in 1 mmol/L, 0.1 mmol/L, and 5 mmol/L treatments, respectively, compared to that in the control. In both the early brown and the early red stage, higher concentration of ACC above a certain level (e.g. 5 mmol/L) resulted in a decreased effect of ACC, thereby indicating deviation from the optimum.

Again, it was noted that the effect of ACC on the stimulation of astaxanthin biosynthesis was quite striking, not in the brown stage, but in the early red stage. Taken together, these results suggest that ACC acts in a dose-dependent as well as stage-dependent manner on astaxanthin accumulation. The microscopic images of H. pluvialis cells under ACC treatment also supported our notion regarding the effect of ACC on either the early brown or the early red stage (Fig. 2G and H).

The study by Gao and Meng (2007) is the only study performed with regards to astaxanthin production using ethylene or ACC treatment. However, this study simply focused on the possible positive effect of ethylene on the vegetative stage of H. pluvialis for astaxanthin biosynthesis, without considering the different effect of ACC that is dependent on the cell stage of H. pluvialis, as presented in this manuscript. Therefore, our data

showing the different outcomes of ACC treatment on different cell stages suggest for the first time that caution is needed when applying either ethylene or ACC as a mimic of ethylene treatment. Since nutrient as well as light penetration in the algal suspension differs in different stages of H. pluvialis, it is also reasonable to consider the influence of multiple factors associated with the cell stage of H. pluvialis. Particularly, the nutrients necessary for growth must be more available at the brown stage than those at the red stage, which might in turn result in difference in the extent of ACC effect on astaxanthin production. We speculated that the effect ACC treatment could stimulate the growth of H. pluvialis in part, due to the availability of nutrients at the brown stage. On the other hand, ACC effect could be more biased toward astaxanthin production at the red stage, because of limited availability of nutrients. Further study might be necessary to elucidate the exact effect of ACC either at brown or red stage of H. pluvialis.

Furthermore, in plants, the impact of exogenous ethylene on carotenoid accumulation has been demonstrated to appear paradoxically between species, either as upregulation (Rodrigo & Zacarias, 2007; Stewart & Wheaton, 1972) or as downregulation (Barreto et al., 2011; Kang & Burg, 1972). Likewise, it is evident that the response to ethylene or ACC must vary due to the dissimilarity between microalgal organisms, as well as between cellular states and external conditions.

Chlorophyll is generally downregulated during cyst formation (Danxiang et al., 2013). However, in our study, chlorophyll content was observed to increase with increasing concentrations of ACC, while astaxanthin responded in a dose-dependent manner. Therefore, a crosstalk between astaxanthin synthesis and photosynthesis might exist (Danxiang et al., 2013) but not an inverse relationship. This view is supported by the

findings of a recent study which showed an increment in both chlorophyll and astaxanthin in H. pluvialis cells after exposure to methyl jasmonate (Raman & Ravi, 2011).

3.3. Distinct patterns in transcription of BKT and CHY upon ACC treatment BKT and CHY are two genes that are involved in the final steps of astaxanthin biosynthesis in H. pluvialis (Grünewald et al., 2000; Steinbrenner & Linden, 2001; Vidhyavathi et al., 2008). Figure 3 shows an upregulation in the expression of BKT and CHY expression at the brown and red stages upon ACC treatment. For the brown stage, BKT transcription was increased in accordance with the ACC concentration, where at 5 mmol/L, it was 11-fold higher than the untreated control after 15 days of ACC treatment (Fig. 3A). A similar trend was observed in the treatment at the red stage, where the influence of 1 mmol/L ACC was comparable to that of 5 mmol/L ACC and BKT transcription was roughly 3-fold higher than the control after 15 days of ACC treatment (Fig. 3B). However, it appears that there is no exact correlation between the transcription levels of BKT and astaxanthin content. On the other hand, CHY expression at both stages was in strong agreement with the amount of astaxanthin, representing a dose-dependent response. The highest transcription level was observed with 1 mmol/L ACC treatment, which showed 2.1-fold higher expression than the control in the brown, encystment stage (Fig. 3C), and up to 21-fold higher expression in the red, cyst stage after 15 days of ACC treatment (Fig. 3D). All treatments at the red stage showed substantial increases in transcription levels. Identical patterns of expression between the transcripts of CHY and the astaxanthin concentrations indicate that astaxanthin biosynthesis may be under the direct control of CHY at the transcriptional level.

Methyl jasmonate and gibberellin A3 was reported to upregulate BKT transcription in H. pluvialis but in a different pattern from astaxanthin accumulation (Lu et al., 2010). A study in Arabidopsis stated that in addition to transcript abundance, other regulatory processes also play a key part in the metabolic adjustments that occur during treatment (Kaplan et al., 2007). Our results suggest that ethylene might directly affect BKT at the mRNA level, while other factors might be involved in regulating the transcription of CHY, which in turn is linked with astaxanthin biosynthesis. In the astaxanthin biosynthetic pathway, it is wellknown that CHY is more closely associated with astaxanthin production than BKT (Grünewald et al., 2000; Steinbrenner & Linden, 2001; Vidhyavathi et al., 2008). In agreement with these previous reports, our data also suggests that the effect of ACC on astaxanthin production must be mediated by the gene expression of both CHY and BKT. However, the influence of ACC is more pronounced on CHY expression than on BKT expression.

3.4. Downregulation of genes encoding antioxidant enzymes upon ACC treatment

ROS (peroxides, superoxide, hydroxyl radical, and singlet oxygen) are known to be cytotoxic at large amounts (Apel & Hirt, 2004). Their amount is thus controlled by scavenging mechanisms and by enzymatic or non-enzymatic accumulation of secondary metabolites like astaxanthin to quench this toxicity (Apel & Hirt, 2004). Our study revealed a reduction in the transcription levels of CAT and SOD, which encode ROS-detoxifying enzymes, upon ACC treatment at both the brown and the red stages (Fig. 4A and 4B). Under high light conditions, the activities of CAT and SOD among antioxidant enzymes, were found to be decreased whereas astaxanthin in H. pluvialis was massively enhanced

(Park et al., 2008). It was then suggested that astaxanthin might involve a non-enzymatic mechanism, largely replacing the antioxidant enzymes in protecting the red cyst cells against high light stress. In accordance with this finding, our data also suggested the opposite relationship between antioxidant enzymes and astaxanthin production (Fig. 4). Further studies might be necessary to elucidate the mechanism affected by ethylene or ACC in ROS-mediated cell signaling.

3.5. Effect of ACC on astaxanthin production under different light intensities

Since astaxanthin production is greatly influenced by light intensity (Fábregas et al., 2000; Giannelli et al., 2015; Hagen et al., 2001; Park et al., 2008), we next examined the compounding effect of ACC along with light intensity. We confirmed that high irradiation helps in promoting astaxanthin accumulation, and the simultaneous application of ACC (1 mmol/L) with irradiation showed an inducing influence, regardless of light intensity (Fig. 5). However, whereas the effects were evident at 20 and 40 µmol m-2 s-1, ACC only exhibited a marginal stimulatory effect at 80 µmol m-2 s-1. The microscopic observations also supported the alteration of H. pluvialis cells according to the extent of astaxanthin level upon different light intensities with ACC treatment (data not shown). The results indicated that the synergistic effect of irradiation and ACC could occur under lower light intensity. However, an increase in light intensity eventually leads to override of light intensity on astaxanthin production, masking the effect of ACC. Increased ethylene production has been widely reported under high light intensity and CO2 sufficiency in plants (Grodzinski et al., 1983; Yang & Hoffman, 1984) and in the cyanobacterium Synechocystis (Ungerer et al., 2012). Besides, in the previous section, we also found

retarded astaxanthin formation upon exposure to high ACC concentration (i.e. 5 mmol/L). Taken together, our data suggest that there might be excessive conversion of ACC to ethylene under high light conditions, resulting in an inhibitory effect on astaxanthin biosynthesis. This speculation interestingly might be advantageous in practical applications whenever light and ACC are simultaneously applied. The data suggested that only a small amount of ACC could be enough for a stimulatory effect on astaxanthin production under high irradiation. In another perspective, the astaxanthin amount yielded via the application of 1 mmol/L ACC under 40 µmol m-2 s-1 was almost equivalent to that obtained under 80 µmol m-2 s-1. Thus, we speculate that with simultaneous application of ACC and irradiation, a relatively lower light illumination could be employed for a similar extent of astaxanthin production, possibly saving the cost of the light energy used for astaxanthin production.

3.6. Establishment of the two-stage application of ACC for astaxanthin production

Previously, we demonstrated that ACC treatment in the vegetative stage of H. pluvialis resulted in increased astaxanthin production via a positive influence of growth (data submitted for a publication). In this study, we further demonstrated the positive effect of ACC on the late growth stage of either the early brown or the early red stage. Considering the previous observation and the current data in this study, it is conceivable that if ACC were applied gradually at different time points in H. pluvialis growth, further enhancement in biomass as well as astaxanthin productivity could be achieved. In order to test our speculation, we proposed a novel strategy involving two-stage application of ACC. To test our strategy, ACC was supplemented at two different time points in H. pluvialis growth; (1) ACC supplementation at the vegetative stage to increase the growth of H. pluvialis, and (2)

at the early cyst stage to enhance astaxanthin production in H. pluvialis. Two different doses of ACC (0.1 mmol/L or 1 mmol/L) were chosen and ACC was treated either once in the initial cultivation or twice complying with the two-stage application of ACC. Consistent with our speculation for a novel two-stage approach, there was a significant increase in astaxanthin production with the employment of ACC supplementation at the vegetative and the early cyst stage, compared to the control without any ACC supplementation (Fig. 6). By the two-stage application of ACC at a dosage of 0.1 mmol/L, astaxanthin content could be increased by 2-fold than the control (Fig. 6). Similar trends were observed with ACC used at 1 mmol/L in two-stage applications. However, even though astaxanthin content could be further enhanced increase in the applied ACC concentration up to 1 mmol/L, the increase in astaxanthin content was not as high compared to the 0.1 mmol/L dosage. Therefore, our results indicated that two-stage application of ACC is effective even at a lower concentration of 0.1 mmol/L.

In summary, we proposed a following process model for the two-stage application of ACC in H. pluvialis cultivation (Fig. 6B). As illustrated in Fig. 6B, there must be a significant difference between cells untreated with ACC (the control) and the two-stage application of ACC. In the control, the initial stage of H. pluvialis shows two flagella and is gradually transformed to the resting cyst stage, which is usually accompanied with the accumulation of a thick cell wall as well as with astaxanthin biosynthesis. On the other hand, overall, the process of both H. pluvialis growth and astaxanthin production could be significantly enhanced by the two-stage application of ACC. 1. As the first stage treatment, ACC treatment (0.1 mmol/L) at the vegetative stage increases the biomass productivity of H. pluvialis by promoting cell division. 2. Increases in cell division facilitate the transformation of H. pluvialis cells to the early cyst stage. 3. As the second stage treatment,

ACC supplementation (0.1 mmol/L) at the early cyst stage resulted in accelerated accumulation of astaxanthin in H. pluvialis.

Taken together, our novel approach with the two-stage application of ACC could open a new possibility to facilitate both biomass and astaxanthin production in H. pluvialis.

4. Conclusions

Our study revealed the effect of ACC on different cell growth stages of H. pluvialis. Though the effect of ACC was dependent on dosage, astaxanthin production was significantly enhanced by ACC treatment at the early red stage. The analyses for the transcriptional abundance of the related genes also supported the notion of enhanced astaxanthin biosynthesis after ACC treatment. The effect of ACC under the different light intensities also indicated the possible application of ACC in combination with light. Finally, we proposed a novel two-stage application system for ACC to increase biomass productivity as well as astaxanthin production.

Acknowledgements

This research was financially supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2014R1A1A2055300).

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Figure Captions

Figure 1. Growth curve of Haematococcus pluvialis. The arrows represent time points at which ACC was added. Microscopic images of H. pluvialis cells at the early encystment stage (B) and the early cyst stage (C), when ACC treatment was applied.

Figure 2. Effects of ACC on chlorophyll content (A) and (B); biomass production (C) and (D); astaxanthin production (E) and (F), with ACC added at the brown stage and red stage, respectively. (G) and (H) Microscopic images of H. pluvialis cells being treated with different ACC concentrations either at the brown (G) or the red stage (H).

Figure 3. Effects of ACC on transcriptional levels of genes related to astaxanthin synthesis. (A) and (B) represent the transcriptional levels of BKT after ACC treatment at the brown and the red stage, respectively; (C) and (D) represent the transcriptional levels of CHY after ACC treatment at the brown and the red stage, respectively. After 1, 5 and 15 days later after the ACC treatment, the expressions of individual gene were measured.

Figure 4. Effects of ACC on transcriptional levels of genes related to astaxanthin synthesis. (A) and (B) represent the transcriptional levels of CAT after ACC treatment at the brown and the red stage, respectively; (C) and (D) represent the transcriptional levels of SOD after ACC treatments at the brown and the red stage, respectively. After 1, 5 and 15 days later after the ACC treatment, the expressions of individual gene were measured.

Figure 5. Effects of ACC under different light intensities on astaxanthin production (“E” stands for “µmol m-2 s-1”). ACC exhibited significant stimulatory effects under 20 and 40 µmol m-2 s-1 but less remarkable effects under 80 µmol m-2 s-1.

Figure 6. A novel two-stage application system for ACC to increase both biomass productivity and astaxanthin production. (A) Astaxanthin contents after two-step ACC treatment. ACC was first added to vegetative cells to increase their growth, and the second treatment was applied when cells were at the early red stage. ACC increased astaxanthin content compared to the control and the two-step treatment further enhanced the astaxanthin content. (B) The schematic illustration for the process of the two-stage application of ACC. First, ACC treatment (0.1 mmol/L) at the vegetative stage increases the biomass productivity of H. pluvialis by promoting cell division. Second, ACC supplementation at the early cyst stage (0.1 mmol/L) accelerates the accumulation of astaxanthin in H. pluvialis.

Tables

Table 1. Primer sequences specific for BKT, CHY, CAT, SOD, and Actin genes. Actin was used as the endogenous control.

Table 1. Target

Primer

Nucleotide sequence (5’-3’)

Hbkt4F1

5’-GGCACTAATGGTCGAGCAGAA-3’

Hbkt4R1

5’-ACAATGAAGACTGCGGCGATG-3’

Hcrt1F1

5’-CATTGCCATCTTCGCCACCTA-3’

Hcrt1R1

5’-GGGCAGTCCATTGGATGATTGG-3’

Hsod2F1

5’-GCCACTGGAACCACAGCTTTTTC-3’

Hsod2R1

5’-ATCCTTTCGCAGCAAAGCCTGCA-3

HcatF1

5’-GAAGTCACGCATGATGTCAGCCA-3’

HcatR1

5’-ATCCAGCAAGGAATGGGTACCAG-3

HactF2

5’-CTCAGCGTTTAGCCTTGTCTG-3’

HactR2

5’-GCCATTGACAAGGAGTTCACG-3’

BKT

CHY

SOD2

CAT

Actin

A)

B)

C)

Figure 1. Growth curve of Haematococcus pluvialis. The arrows represent time points at which ACC was added. Microscopic images of H. pluvialis cells at the early encystment stage (B) and the early cyst stage (C), when ACC treatment was applied.

A)

B)

C)

D)

E)

F)

Figure 2. Effects of ACC on chlorophyll content (A) and (B); biomass production (C) and (D); astaxanthin production (E) and (F), with ACC added at the brown stage and red stage, respectively. (G and H) Microscopic images of H. pluvialis cells being treated with different ACC concentrations either at the brown (G) or the red stage (H).

G)

H)

Figure 2. Effects of ACC on chlorophyll content (A) and (B); biomass production (C) and (D); astaxanthin production (E) and (F), with ACC added at the brown stage and red stage, respectively. (G and H) Microscopic images of H. pluvialis cells being treated with different ACC concentrations either at the brown (G) or the red stage (H).

18

A)

Relative transcriptional abundance

Relative transcriptional abundance

20 Control 0.1 m M 1 mM 5 mM

16 14 12 10 8 6 4 2 0 0

5

3.5 3.0

Control 0.1 mM 1 mM 5 mM

C)

2.5 2.0 1.5 1.0 0.5 0.0 0

15

5

day

35

B)

Control 0.1 m M 1 mM 5 mM

25 20 15 10 5 0 0

5

15

day

Relative transcriptional abundance

Relative transcriptional abundance

day

30

15

40

D)

Control 0.1 mM 1 mM 5 mM

30

20

10

0 0

5

15

day

Figure 3. Effects of ACC on transcriptional levels of genes related to astaxanthin synthesis. (A) and (B) represent the transcriptional levels of BKT after ACC treatment at the brown and red stage, respectively; (C) and (D) represent the transcriptional levels of CHY after ACC treatment at the brown and red stage, respectively.

A)

Control 0.1 m M 1 mM 5 mM

1.0

0.8

0.6

0.4

0.2

0.0 0

5

Relative transcriptional abundance

Relative transcriptional abundance

1.2

15

1.4 1.2

Control 0.1 mM 1 mM 5 mM

C)

1.0 0.8 0.6 0.4 0.2 0.0 0

5

day 1.2

1.4 1.2

Control 0.1 mM 1 mM 5 mM

B)

1.0 0.8 0.6 0.4 0.2

Relative trancriptional abundance

Relative transcriptional abundance

day

15

1.0

Control 0.1 mM 1 mM 5 mM

D)

0.8

0.6

0.4

0.2

0.0

0.0 0

5

15

day

0

5

15

day

Figure 4. Effects of ACC on transcriptional levels of genes related to antioxidant enzymes. (A) and (B) represent the transcriptional levels of CAT after ACC treatment at the brown and red stage, respectively; (C) and (D) represent the transcriptional levels of SOD after ACC treatment at the brown and red stage, respectively.

Figure 5. Effects of ACC under different light intensities on astaxanthin (“E” stands for “µmol m-2 s-1”). ACC exhibited significant stimulatory effects under 20 and 40 µmol m-2 s-1 but less remarkable effects under 80 µmol m-2 s-1.

A)

B) No ACC Two-stage ACC treatment ACC(I)

ACC(II)

Figure 6. A novel two-stage application system for ACC to increase both biomass productivity and astaxanthin production. (A) Astaxanthin contents after two-step ACC treatment. ACC was first added to vegetative cells to increase their growth, and the second treatment was applied when cells were at the early red stage. ACC increased astaxanthin content compared to the control and the two-step treatment further enhanced the astaxanthin content. (B) The schematic illustration for the process of the two-stage application of ACC. First, ACC treatment (0.1 mmol/L) at the vegetative stage increases the biomass productivity of H. pluvialis by promoting cell division. Second, ACC supplementation at the early cyst stage (0.1 mmol/L) accelerates the accumulation of astaxanthin in H. pluvialis.

Target

Primer

Nucleotide sequence (5’-3’)

BKT

Hbkt4F1

5’-GGCACTAATGGTCGAGCAGAA-3’

Hbkt4R1

5’-ACAATGAAGACTGCGGCGATG-3’

Hcrt1F1

5’-CATTGCCATCTTCGCCACCTA-3’

Hcrt1R1

5’-GGGCAGTCCATTGATGATTGC-3’

Hsod2F1

5’- GCCACTGGAACCACAGCTTTTTC-3’

Hsod2R1

5’- ATCCTTTCGCAGCAAAGCCTGCA-3’

HcatF1

5’- GAAGTCACGCATGATGTCAGCCA-3’

HcatR1

5’- ATCCAGCAAGGAATGGGTACCAG-3’

HactF2

5’-CTCAGCGTTTAGCCTTGTCTG-3’

HactR2

5’-GCCATTGACAAGGAGTTCACG-3’

CHY

SOD2

CAT

Actin

Table 1. Primer sequences specific for BKT, CHY, CAT, SOD, and Actin genes.

Highlights: -

The effects of ACC on different cell stages of H. pluvialis were investigated.

-

Astaxanthin production could be increased whenever the cells were treated at the early red stage.

-

Transcriptional levels of relevant genes supported enhanced astaxanthin biosynthesis.

-

Synergistic effect of ACC and light intensity was also confirmed.

-

Finally, two-step applications of ACC were proposed and verified.