A strategy for promoting astaxanthin accumulation in Haematococcus pluvialis by 1-aminocyclopropane-1-carboxylic acid application

Accepted Manuscript Title: A strategy for promoting astaxanthin accumulation in Haematococcuspluvialis by 1-aminocyclopropane-1-carboxylic acid application Author: Changsu Lee Yoon-E Choi Yeoung-Sang Yun PII: DOI: Reference:

S0168-1656(16)31468-7 http://dx.doi.org/doi:10.1016/j.jbiotec.2016.08.012 BIOTEC 7652

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Journal of Biotechnology

Received date: Revised date: Accepted date:

29-3-2016 2-8-2016 16-8-2016

Please cite this article as: Lee, Changsu, Choi, Yoon-E, Yun, Yeoung-Sang, A strategy for promoting astaxanthin accumulation in Haematococcuspluvialis by 1-aminocyclopropane-1-carboxylic acid application.Journal of Biotechnology http://dx.doi.org/10.1016/j.jbiotec.2016.08.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

A strategy for promoting astaxanthin accumulation in Haematococcuspluvialis by 1aminocyclopropane-1-carboxylic acid application

Changsu Lee1, Yoon-E Choi2,*, Yeoung-Sang Yun1,*

Department of Bioprocess Engineering, Chonbuk National University, Jeonju 54896, Korea1 Division of Environmental Science & Ecological Engineering, Korea University, Seoul 02841, Korea2

*Corresponding author to Yoon-E Choi, [email protected], or to Yeoung-Snag Yun, [email protected]

Highlights: - The effect of ACC, a precursor of ethylene, to promote astaxanthin was examined - ACC could enhance the growth of H. pluvialis, thereby promoting astaxanthin accumulation - Gene expressions related with astaxanthin biosynthesis were thoroughly examined - Cellular extent of reactive oxygen species (ROS) were monitored in response to the treatment of ACC - A direct treatment of ethylene originated from banana peels verified the effect of ACC

ABSTRACT The green algae Haematococcuspluvialis is a freshwater unicellular microalga belonging to Chlorophyceae. It is one of the best natural sources of astaxanthin, a secondary metabolite commonly used as an antioxidant and anti-inflammatory agent. Due to the importance of astaxanthin, various efforts have been made to increase its production. In this study, we attempted to develop a strategy for promoting astaxanthin accumulation in H. pluvialis using 1-aminocyclopropane-1-carboxylic acid (ACC), a precursor of ethylene (normally known as an aging hormone in plants). Our results demonstrated that ACC could enhance the growth of H. pluvialis, thereby promoting astaxanthin accumulation. Therefore, ACC has an indirect influence on astaxanthin production. We further verified the effect of ACC with a direct treatment of ethylene originated from banana peels. These results indicate that ethylene could be applied as an indirect method for enhancing growth and astaxanthin biosynthesis in H. pluvialis. Keywords: Haematococcuspluvialis; ACC; growth; astaxanthin; ROS. 1. INTRODUCTION The astaxanthin is famous for several immunological, as well as neuroprotective, potentials. Due to the various protective effects of astaxanthin, it is beneficial for human health in potentially preventing various diseases (Guerin et al., 2003). Astaxanthin is predominantly present in a broad range of seafood, such as shrimp, lobster, salmon, red seabream, trout, and fish eggs. However, animals naturally containing astaxanthin do not have the ability to biosynthesize astaxanthinthemselves, therefore they must obtain astaxanthin from environmental sources, such as microalgae (Orosa et al., 2005). Currently, the microalga Haematococcuspluvialis is known to have the highest content of astaxanthin. Commercially, astaxanthin accumulates more than 30 g from kg-1 of H. pluvialisdry biomass (Guerin et al., 2003; Olaizola and Huntley, 2003).

H. pluvialis has a unique life cycle, varying markedly in color and cell size. Under favorable conditions, H. pluvialis maintains a green motile stage, with two flagella actively undergoing cell divisions. This green stage could be categorized as the vegetative growth stage with lower accumulation of astaxanthin (Norihiko et. al., 2001; Tomohisa et. al., 2006). However, upon unfavorable environmental conditions, H. pluvialiscells are dramatically transformed into a resting phase, known as a cyst. At that stage, H.pluvialiscells completely stop cell division and accumulate a thick, resistant cell wall (Boussiba, 2000; Kakizono et al., 1992). The biosynthesis of astaxanthin in H.pluvialisisstrictly limited to the cyst stage, thereby coping with any oxidative stresses generated from a wide variety of unfavorable environments (Choi et al., 2002; Kang et al., 2007). Therefore, the accumulation of astaxanthin in H. pluvialis is induced by various stress conditions. Factors inducing astaxanthin production include a wide variety of unfavorable environmental stimuli such as high light intensity, pH, salinity, high temperature, nitrogen limitation, and phosphate deficiency (Boussiba and Vonshak, 1991; Fábregas et al., 2001; Kang et al., 2005; Sarada et al., 2002). However, there is still much room for improvement in the extent of astaxanthin production by customizing the conditions conducive for astaxanthin biosynthesis. To be economically feasible, various efforts have increased astaxanthin content by establishing the most effective inductive conditions for astaxanthin biosynthesis. While researchers have invested substantial efforts to develop the most effective ways to enhance astaxanthin production, our protocol using H. pluvialisis far from complete. Therefore, limited information is available on the microalgal biotechnological process of astaxanthin production. Ethylene is a small hydrocarbon gas, which is a product of combustion in the chemical industry, and an important natural plant hormone with a wide range of effects in plants, thus it

is widely used in agriculture. The applications of ethylene include seed germination, abscission, flower senescence, and fruit maturation (Johnson and Ecker, 1998; Wang et al., 2002). Particularly, ethylene acts as an aging hormone in plants, causing fruit to ripen. Due to its crucial roles as a regulator in fruit ripening and flower senescence, its production must be tightly regulated by developmental and environmental factors. Therefore, throughout the life of the plant, ethylene production is induced only under the certain stages of growth, implying that the development and growth of the plant could be significantly altered by the artificial application of ethylene. So far, much information has been accumulated concerning the biosynthesis of ethylene. The precursor molecule for ethylene biosynthesis is the amino acid methionine, which is modified to S-adenosyl-L-methionine (SAM) by the enzyme Met Adenosyltransferase. Subsequently, SAM is then converted to 1-aminocyclopropane-1-carboxylic acid (ACC) by the key enzyme ACC synthase (ACS) (Wang et al., 2002). Therefore, ACC is the indispensable molecular precursor of ethylene, directly triggering the biosynthesis of ethylene. Despite ethylene’s central role in accelerating the senescence of plants, limitedinformation is available on the functions of the ethylene signaling in microalgal species. Since astaxanthin biosynthesis could be activated only with the onset of the senescence of H. pluvialiscells, it is reasonable to suppose that ethylene might enhance astaxanthin production. Despite the fact that ethylene could be a factor in astaxanthin production, little study has attempted to apply it in astaxanthin production using H. pluvialis. In this study, we utilized ACC, an indispensable precursor of ethylene, as a key factor to promote the astaxanthin biosynthesis. Since ACC has a similar effect to that of ethylene, and is converted to ethylene via enzymatic transformation, we hypothesized that exogenous applications of ACC may have a similar response as ethylene in plants. Particularly, we

expected a senescence of H. pluvialis cells after introducing ACC, which in turn could lead to enhanced astaxanthin production. Interestingly, however, ACC exhibited enhanced the growth of H. pluvialis, thereby promoting astaxanthin accumulation. Therefore, ACC indirectly influenced astaxanthin production. We further verified the effect of ACC with the application of ethylene gas originating from banana peels in a closed culture system of H. pluvialis. Our data clearly indicated that ethylene has a positive effect on the growth and biosynthesis of astaxanthin in H. pluvialis, thus suggesting a potential application of ethylene in microalgae biotechnology, particularly using H. pluvialis. 2. Materials and methods 2.1. Strain and culture conditions Unicellular green alga,H.pluvialis (UTEX# 2505) was obtained from the algae culture collection at the University of Texas, Austin, Texas, USA. Strains were regularly cultivated in a 250-mL round-bottomed flask with 150 mL of sterile OHM medium (Fábregas et al., 2000) at 25 °C. Continuous illumination was supplied at an average light intensity of 50 μmol m-2 s-1 and aeration was supplied at 1 vvm of air after filtering through 0.2 μm syringe filter. To test the effect of -aminocyclopropane-1-carboxylic acid (ACC) on H.pluvialisbiology, as well as astaxanthin production, various amounts of ACC were applied to the culture of H. pluvialis. Treated concentrations of ACC were 0, 0.1, 0.2, 0.5, 1 mM, respectively. After seven days of cultivations, microalgal biomass was harvested and analyzed for the extent of astaxanthin content.

2.2. Astaxanthin measurement

The astaxanthin concentration was measured using spectrophotometer (T60 U, Korea). To analyze astaxanthin, cells were harvested by centrifugation for 10 min at 13,000 rpm, washed twice in de-ionized (DI) water, and then homogenized in 90 % acetone using beadbeater (MBB-16, USA). After centrifugation at 13,000 rpm for 1 min, supernatants were measured based on optical density at the wavelength of 474 nm, and calculated using astaxanthin correlation graph with HPLC. The astaxanthin standard (013-23051, Wako) was used for calibration. The content of astaxanthin production (mg L-1) was calculated using the following equation: Astaxanthin = (OD474 − 0.0831)/0.1426 To further verify the amount of astaxanthin, HPLC analyses were also further employed. HPLC was carried out using a Shimadzu LC (Shimadzu, Kyoto, Japan) system, equipped with a CBM-20A system controller, a LC-20AD pump, a DGU-20A3R degasser, and a SPD20A UV-vis detector. The chromatographic conditions were YMC-Carotenoid column (4.6 ×250mm, 5 μm), and the mobile phase used for gradient elution was composed of methanol as system A, t-butylmethylether as system B, and 1% phosphoric acid aqueous as system C. The progress of HPLC was monitored at 474 nm.

2.3. Growth measurements H. pluvialis growth was determined by direct counting with a hemocytometer (Hausser Scientific, Horsham, PA) using an OPTINITY microscope (KB-500, Korea). The dry cell weight (DCW) was measured by filtering the algal suspension through a pre-dried and preweighed, 0.45 μm cellulose nitrate membrane filter (Whatman, USA) and drying in an oven

at 80°C for 24 h. The specific growth rate (μ) was calculated based on the following equation: μ = (lnX1 – lnX0/t1 – t0) where X1 and X0 were the biomass concentration (g L-1) at time t1 and t0, respectively (Kim et al., 2014). The morphology of H. pluvialis was measured with OPTINITY microscope camera (C30, Korea) from more than one hundred microalgal cells, randomly selected.

2.4. Transcription analysis Total RNA was extracted from 100 mg of H. pluvialis culture with TRIzol reagent (Invitrogen) according to the manufacturer’s protocols.Nucleic acid concentrations were measured spectrophotometrically at 260 nm. The integrity and purity of the RNA were determined by 260/280 nm ratios and separated by electrophoresis on a 1% agarose gel. cDNA was synthesized with a capacity RNA-to-cDNA kit (Applied Biosystems). An enzyme mixture of 5 μg of total RNA and 2X RT was mixed in a reaction tube and incubated at 37°C for 1 h, and then inactivated 95°C for 5 min, after quickly cooled on ice. Three genes related to astaxanthin biosynthesis, and one related to chlorophyll in H. pluvialis, were selected as target genes. These select genes were: (i) BKT (beta-carotene ketolase) (NCBI GeneID: D45881), (ii) CHY (carotenoid hydroxylase) (NCBI GeneID: AF162276), (iii) LCY (lycopene beta cyclase) (NCBI GeneID: AY182008), (iv) Cbr (carotene biosynthesis-related protein) (NCBI GeneID: AY878537), cue for chlorophyll a-b binding protein. QRT-PCR analysis was carried out with SYBR-Green® as the fluorescent. Gene specific primers for BKT gene (Hbkt4F1 and Hbkt4R1), CHY gene (Hcrt1F1 and Hcrt1R1), LCY gene (Hlbc1F1 and Hlbc1R1), and Cbr (Hcbr1F1 and Hcbr1R1) were used

for giving rise to amplicons. The expression of each gene was normalized to endogenous actin (NCBI GeneID: DV203941) gene expression with specific pair of primers (HactF2 and HactR2).

2.5. ROS analysis In situ detection of the superoxide radical was performed by algae cultures with nitroblue tetrazolium (NBT) (N6876, Sigma–Aldrich) staining following the protocol described previously (Rao and Davis, 1999). Three biological replications were performed. Cultures grown in OHM media for 7 days were centrifuged and re-suspended in 0.2% NBT. After incubation for 12 h, cells were re-centrifuged and re-suspended in 10 ml of 50% glacial acetic acid. For quantification, a bead-beater was used to break the stained cells, and the extent of ROS was measured with the absorbance at 560 nm (Kim et al., 2014).

2.6. Direct application of ethylene with banana peels Cells were regularly cultivated in 250 mL round-bottomed flasks with 150 mL of sterile OHM medium at 25°C. Continuous illumination was supplied at an average light intensity of 50 μmol m-2 s-1, and aeration was supplied at 1 vvm of air after filtering through 0.2 μm syringe filter. To apply ethylene directly, banana peels was put in a closed container with the only possible outlet leading to microalgal cultivation (Fig. 5A). Banana peels were treated as either low weight (429g) or high weight (1,048g). More than three biological replications were performed to obtain standard deviations.

3. Results and discussion 3.1. Effect of ACC treatment to promote the growth rate of H. pluvialis Ethylene is a well-known hormone that promotes the maturation and aging of fruit in plants. We hypothesized that the activity of ethylene could be a powerful inducer of astaxanthin accumulation from H. pluvialis, since the maturation of H. pluvialis cells is tightly linked with astaxanthin biosynthesis. However, in general, ethylene is difficult to control, because this hormone primarily exists in the gaseous stage. Thus, we turned to ACC as an indispensable precursor of ethylene, and adjusted various concentrations of ACC. We speculated that the biosynthesis of astaxanthin in H. pluvialis would depend on the various ACC concentrations (0, 0.1, 0.2, 0.5, 1 mM). However, in contrast, we observed different effects of ACC from our initial expectation. Interestingly, our results showed that ACC promoted H. pluvialisgrowth. Therefore, unexpectedly, ACC had an effect not only on cell senescence or subsequent astaxanthin biosynthesis, but on the growth of H. pluvialis. Proportionate to the treated concentrations of ACC, H. pluvialiscells clearly displayed increased growth. Particularly, cell density of H. pluvialis with 1 mM ACC showed 2.4-fold higher growth than the non-treated control after seven days of incubation from the inoculums (Fig. 1). In addition, in the initial stage of cultivation, the amount of chlorophyll increased proportionate to the concentration of ACC (data not shown). These results clearly suggest that the ACC is directly related to the growth of H. pluvialis. Higher concentration of ACC up to 5 mM was also tested and higher application of ACC (1 mM to 5 mM) led to the opposite results helping maintaining the green stage instead (data not shown). Our data is consistent with the previous report suggesting the optimum concentration of ACC exists in microalgal species (Patrick, M. et. al., 1993).

3.2. ACC has an indirect effect on the production of astaxanthin. ACC turned out to have an effect on promoting H. pluvialis growth consistently. And the increased growth via the ACC applications eventually helped increase the accumulation of astaxanthin (Fig. 1 and Fig. 2C). The astaxanthin content also increased with the treatment concentrations of ACC. Particularly, an increase from 0 mM to 1 mM ACC resulted in a rapid change in astaxanthin pigmentation, which can be observed with the naked eye (Fig 2A). Microscopic images also clearly revealed an enhancement of astaxanthin biosynthesis with relation to ACC treatment (Fig. 2B). We quantified the extent of astaxanthin, based on certain cell numbers and revealed that astaxanthin accumulation could increase approximately 1.8-fold higher under the 1 mM treatment, compared to that of non-treated cells (Fig. 2C). In general, increased growth in H. pluvialis led to earlier cell senescence by accelerating cell maturity. Since the onset of astaxanthin biosynthesis in H.pluvialisis strictly linked with cell senescence (Domınguez-Bocanegra et al., 2004; Sarada et al., 2002), it is likely that the effect of ACC to astaxanthin biosynthesis must originate from the increased cell growth. Therefore, our results suggested that ACC influences the growth of H. pluvialis, which in turn indirectly leads to enhanced astaxanthin production. The effect of ACC is, not directly, but indirectly related to the astaxanthin production in H. pluvialis.

3.3. ROS analysis with ACC treatment Reactive oxygen species (ROS) are important, highly reactive oxygen containing compounds. ROS can be generated as a natural byproduct of regular normal cellular metabolism. However, ROS levels also can be differentially regulated in response to

various environmental stresses. In either case, ROS have a key functional role as messengers for cellular metabolism across many living species. Therefore, the level of ROS must be tightly up-regulated or down-regulated for the proper cellular responsiveness to either favorable or unfavorable environmental cues. A sudden increase in ROS in response to the oxidative stress in cellular system can lead to substantial damage to cells. Microalgae, including H. pluvialis,are also responsive to environmental cues producing different ROS levels, and therefore need to cope with sudden oxidative stress from various environmental stresses. To detoxify the reactive ROS, H. pluvialisis thought to employ two parallel strategies: one is a thick cell wall, and the other is astaxanthin biosynthesis. Owing to its strong ROS quenching ability, astaxanthin is synthesized along with the accumulation of ROS under stress conditions in H. pluvialis (Boussiba, 2000; Fan et al., 1998; Kobayashi et al., 1993). Therefore, the extent of ROS could be an indirect indicator of astaxanthin production in H. pluvialis. Based on that, we measured the extent of ROS upon various ACC treatments. As shown above, ACC has a positive influence on H. pluvialisgrowth, thereby enhancing astaxanthin production (Fig. 1 and Fig. 2). A standard nitroblue tetrazolium (NBT) staining protocol was employed to quantify ROS extent in H. pluvialiscells upon various ACC treatments. As expected, many different levels of ROS were observed depending on ACC treatments (Fig. 3). An opposite tendency in the pattern of ROS level was observed, in that lower and higher concentrations of ACC treatments resulted in an increase and decrease of ROS extent, respectively. Particularly, more than 0.5 mM of ACC led to significant decreases in cellular ROS level (Fig. 3). It is likely that H. pluvialiscells accumulated increased ROS, along with the senescence of cells caused from the enhanced growth under the lower concentrations of ACC treatment (below 0.2 mM). However, above a certain

concentration (> 0.5 mM) of ACC, H. pluvialiscells began to biosynthesize astaxanthin, and accumulated astaxanthin might help decrease ROS level owing to its strong ROS quenching ability. The rapid decrease of ROS above 0.5 mM was matched well with corresponding astaxanthin production, supporting the tight negative relationship between ROS extent and astaxanthin levels (Fig. 2C and Fig. 3). Much evidence suggested the relationship between cellular ROS level and astaxanthin biosynthesis in H. pluvialis. It is possible that the onset of astaxanthin biosynthesis can be regulated by ROS levels via the involvement of several potential enzymes (Aniya and Anders, 1992; Boussiba, 2000; Miller and Claiborne, 1991). Our results also suggested that ACC has a direct effect on the growth of H. pluvialis, thereby causing the high accumulation of ROS. It is likely that H. pluvialiscells begin to biosynthesize astaxanthin accordingly to cope with the certain levels of ROS extent. Further study will be necessary to elucidate the potential mechanism of astaxanthin production associated with cellular ROS level.

3.4. Differential expressions of genes associated with astaxanthin biosynthesis with ACC treatment We also investigated the influence of ACC on the expression of genes associated with astaxanthin biosynthesis. In H. pluvialis, astaxanthin constitutes the majority of astaxanthin as a secondary carotenoid, and mostly exists in esterified forms floating in the extraplastidic lipid vesicles. Astaxanthin biosynthesis is initiated from a secondary carotenoid (Grünewald et al., 2001; Vidhyavathi et al., 2008). The biosynthesis is carried out with sequential biochemical processes involving multiple enzymes for each of the processes. For example, (i) phytoene synthase (PSY) mediates the conversion of geranylgeranyl diphosphate (GGPP) to phytoene; (ii) Subsequently, phytoene synthase (PDS) converts

GGPP to ζ-carotene; (iii) both ζ-carotene desaturase (ZDS) and carotenoid isomerase (CRTISO) are involved in the transformation of ζ-carotene to lycopene; (iv) lycopene cyclase (LCY) transforms lycopene to β-carotene; (v) carotene ketolase (BKT) catalyzes the conversion of either β-carotene to echinenone or echinenone to canthaxanthin; (vi) and in the final step, β-carotene hydroxylase (CHY) converts canthaxanthin to astaxanthin (Steinbrenner and Linden, 2001; Vidhyavathi et al., 2008). Alternatively, zeaxanthin is competitively produced from β-carotene via the involvement of Cbr, possibly suppressing the production of astaxanthin (Lemoine and Schoefs, 2010). To determine the alteration of in vivoastaxanthin biosynthesis with the extent of ACC applications, total RNA was extracted from H. pluvialisbiomass grown under different ACC concentrations. Several genes, e.g. BKT, CHY, LCY, and Cbr, encoding key enzymes for astaxanthin biosynthesis were selected, and their expressions were quantified with qRT-PCR. qRT-PCR analysis revealed that select genes displayed noticeable differences dependent on the level of ACC. The expressions of both BKT and CHY were significantly increased, along with the increase of treated ACC concentration. In particular, CHY transcripts were more than six-fold upregulated with the treatment of 1 mM ACC, compared to those without ACC treatment as a control (Fig. 4). CHY produced the final product of astaxanthin, whereas BKT produced the direct precursor of astaxanthin (Rick et al., 2006), indicating increased astaxanthin production with ACC treatment. However, both LCY and Cbr expressions displayed the opposite pattern with the expression of BKT and CHY upon ACC treatment. Particularly, the expression of LCY increased temporarily at 0.1 mM ACC treatment, and subsequently decreased within the range of 0.2 mM to 1 mM ACC. The reason for the down-regulation of LCY expression might indicate that β–carotene has already accumulated and transformed to astaxanthin at

above 0.2 mM ACC treatment. Consistent with our data, previous study also suggested that the expression of LCY displayed the opposite pattern with those of CHY. Likewise the treatment of ACC in this study, certain concentration of fulvic acid (FA) application led to the differential trends in the gene expressions between LCY and CHY (Zhao et al., 2015), supporting our notion about the pattern of LCY gene expression. In case of Cbr expression, however, there is a consistent decrease of Cbr transcripts with the increase of ACC treatment levels (Fig. 4). In Dunaliella, Cbr encodes a zeaxanthin-binding protein which positively influences the conversion from β-carotene to zeaxanthin (Hsu and Lee, 2012; Levy et al., 1993). Since the biosynthetic pathways to astaxanthin competes with zeaxanthin (Steinbrenner and Linden, 2001; Vidhyavathi et al., 2008), our results suggested a decrease in zeaxanthin production via ACC treatment, thereby increasing astaxanthin biosynthesis with ACC treatment. Taken together, data in this study provide molecular evidence that BKT and CHY are positively regulated, whereas LCY and Cbr are negatively regulated by ACC in H. pluvialis. Consequently, astaxanthin biosynthesis can be influenced by ACC.

3.4. Further verification with gas generated from banana peels suggests possible application To further verify the effects of ACC, we attempted to apply ethylene gas directly to an algal suspension of H. pluvialis. To examine the effect of ethylene, we searched for the most ubiquitous and enriched natural source of ethylene according to previous knowledge, as well as experience. It turned out that banana is abundant in natural materials, with high ethylene enrichment (Abeles et al., 2012; Xiao et al., 2013). Based on that, we speculated that if the gas originating from banana biomass is applied, the effect on H.

pluvialiscultivation would be similar to that of ACC treatment. Since it is always better to not only consider the material, but also its waste product, banana peels were chosen for the natural source of ethylene. Different amounts of banana peels were placed in a sealed acrylic container, with the only possible gas outlet from the banana peels was into the algal suspension of H. pluvialis(Fig. 5A). Banana peels were treated at either low weight (429g) or high weight (1,048g), and designated as E-429 or E-1048, respectively. Consistent with our previous findings, the application of banana peels led to an enhanced growth of H. pluvialis(Fig. 5 D and E). Furthermore, cell morphologies were changed to match the morphological shift of the resting cyst phase of H. pluvialis, thereby increasing astaxanthin content(Fig. 5B and 5C). ROS analysis also revealed a reduction in ROS extent, along with increased astaxanthinproduction via the application of banana peels due to the high activity of astaxanthin as an ROS quencher (Fig. 5F). All of these data were correlated with the amount of banana peels used, suggesting that differences in biomass, as well as astaxanthin content, must originate exclusively from the effects of ethylene gas from the banana peels. Our results provide important evidence that direct application of ethylene could be useful to promote both the growth and astaxanthin content in H. pluvialis. The study from Gao&Meng is the only study so far performed for astaxanthin production using ethylene or ACC. However, the study from Gao&Mengsimply focused on the positive effect of ethylene on astaxanthin biosynthesis without suggesting any supporting evidence. In this study, we clarified that the effect of ethylene, along with a precursor ACC, originates from the enhanced growth, instead of astaxanthin production itself. Furthermore, we suggested the novel application of banana waste on microalgal cultivation, including H. pluvialis, due to its abundance in ethylene gas. Varied gene

expressions, as well as ROS content after ACC treatment, also supported our results on the effect of ACC or ethylene. Overall, this study illustrated the putative effect of ACC or ethylene (Fig, 6). ACC, as a precursor of ethylene, plays a role in the direct promotion of H. pluvialis growth, thereby causing increased astaxanthin production. Our discovery will provide additional options to further enhance astaxanthin production.

4. Conclusion In this study, we demonstrate a way to facilitate the production of astaxanthin in H. pluvialis, using the ACC and ethylene. ACC does not affect the production of astaxanthin directly, but promoted cell growth, thereby increasing astaxanthin production. We further verified the effect of ACC with the direct treatment of ethylene generated from banana peels. Our results will provide evidence for the applications of either ethylene or ACC for enhancing growth and astaxanthin production in H. pluvialis.

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 LEGENDS Fig. 1.Effects of ACC treatment on H. pluvialis. The ACC promoted H. pluvialis cell growth. All experiments were repeated at least three times. Fig. 2.Morphological shift in H. pluvialis in various ACC concentrations.(A) Photo of algal suspension with ACC treatment, (B) Microscopic images of cells in various ACC treatments, (C) Altered astaxanthin production with different ACC treatments. All experiments were repeated at least three times. Fig. 3. Determination of ROS levels with ACC treatment. All experiments were repeated at least three times . Fig. 4. Expression of genes associated with astaxanthin biosynthesis. BKT (beta-carotene ketolase) and CHY (carotenoid hydroxylase) expressions were increased with the increased concentration of ACC. However, LCY (lycopene beta cyclase) and Cbr (carotene biosynthesis-related protein) expressions decreased with treated ACC levels. All experiments were repeated at least three times. Fig 5. Direct application of ethylene using banana peels. (A) Experimental design with a sealed container with different amounts of banana peels. Only possible gas outlets were directed to microalgal suspension, (B) Morphological shift with the application of ethylene gas, (C) Varied astaxanthin concentrations, (D) cell density, (E) biomass production, (F) ROS extent with the application of banana peels. All experiments were repeated at least three times. Fig. 6.Putative scheme of the effect of ACC treatment on H. pluvialis. For H. pluvialis, ACC serves to directly to promote cell growth. Rapidly growing cells with ACC treatment eventually led to increased production of astaxanthin.

TABLE LEGENDS

Table 1.List and description of primers.

Table 1. Target

Primer name

Nucleotide sequence (5ꞌ-3ꞌ)

BKT

Hbkt4F1

5ꞌ-GGCACTAATGGTCGAGCAGAA-3ꞌ

Hbkt4R1

5ꞌ-ACAATGAAGACTGCGGCGATG-3ꞌ

Hcrt1F1

5ꞌ-CATTGCCATCTTCGCCACCTA-3ꞌ

Hcrt1R1

5ꞌ-GGGCAGTCCATTGATGATTGC-3ꞌ

Hlbc1F1

5ꞌ-GCTAGCGACATTGCTTTGACC-3ꞌ

Hlbc1R1

5ꞌ-ACTTAGGAACTTCTCGCCCTC-3ꞌ

Hcbr1F1

5ꞌ-CGAACTTCCTCAGCTCTTCTC-3ꞌ

Hcbr1R1

5ꞌ-CTCCCACAGCTGCAACAAAAC-3ꞌ

HactF2

5ꞌ-CTCAGCGTTTAGCCTTGTCTG-3ꞌ

HactR2

5ꞌ-GCCATTGACAAGGAGTTCACG-3ꞌ

CHY

LCY

Cbr

Actin

Figure

Fig. 1. Growth 1.6 ACC 0 mM ACC 0.1 mM ACC 0.2 mM ACC 0.5 mM ACC 1 mM

1.4

DCW(g/L)

1.2 1.0 0.8 0.6 0.4 0.2 0.0 0

2

4

Day

6

8

Fig. 2. A

C 50

Con

0.1 mM

0.2 mM

0.5 mM

1 mM

Astaxanthin (mg/g)

40

30

20

10

0 0 mM

0.1 mM

0.2 mM

0.5 mM

1 mM

ACC concentration

B

Con

0.5 mM

0.1 mM

1 mM

0.2 mM

Fig. 3. ROS 1.4

Relative cellular abundance

1.2

1.0

0.8

0.6

0.4

0.2

0.0 Con

0.1 mM

0.2 mM

0.5 mM

ACC concentration

1 mM

Fig. 4. BKT

Cbr 1.4

Relative transcriptional abundance

Relative transcriptional abundance

3.0

2.5

2.0

1.5

1.0

0.5

1.2

1.0

0.8

0.6

0.4

0.2

0.0

0.0 Con

0.1 mM

0.2 mM

Con

1 mM

0.1 mM

CHY

1 mM

LCY 2.5

Relative transcriptional abundance

10

Relative transcriptional abundance

0.2 mM

ACC concentration

ACC concentration

8

6

4

2

2.0

1.5

1.0

0.5

0.0

0 Con

0.1 mM

0.2 mM

ACC concentration

1 mM

Con

0.1 mM

0.2 mM

ACC concentration

1 mM

Fig. 5. B

A

Non-treat

C

D

Astaxanthin

Growth rate

Con E-429 E-1048

30

25

-1

Growth rate (day )

Astaxanthin (mg/g)

Banana peel - 1048g

35

50

40

Banana peel - 429g

30

20

20

15

10 10

5

0 0 day

0 1 day

2 day

3 day

4 day

5 day

6 day

ROS 0.7

Relative cellular abundance

0.6

0.5

0.4

0.3

0.2

0.1

0.0 Con

Con

E-429

Ethylene concentration

Day

E

7 day

E-429

Ethylene concentration

E-1048

E-1048

Fig. 6. ACC (precursor of ethylene)

ACC (precursor of ethylene)