Enhanced astaxanthin production by oxidative stress using methyl viologen as a reactive oxygen species (ROS) reagent in green microalgae Coelastrum sp.

Microalgae are known to be a potential resource of high‐value metabolites that can be used in the growing field of biotechnology. These metabolites constitute valuable compounds with a wide range of applications that strongly enhance a bio‐based economy. Among these metabolites, astaxanthin is considered the most important secondary metabolite, having superior antioxidant properties. For commercial feasibility, microalgae with enhanced astaxanthin production need to be developed. In this study, the tropical green microalgae strain, Coelastrum sp., isolated from the environment in Malaysia, was incubated with methyl viologen, a reactive oxygen species (ROS) reagent that generates superoxide anion radicals (O2‐) as an enhancer to improve the accumulation of astaxanthin. The effect of different concentrations of methyl viologen on astaxanthin accumulation was investigated. The results suggested that the supplementation of methyl viologen at low concentration (0.001 mM) was successfully used as a ROS reagent in facilitating and thereby increasing the production of astaxanthin in Coelastrum sp. at a rate 1.3 times higher than in the control.


Introduction
The ketocarotenoid astaxanthin (3,3'dihydroxy4,4' diketoβcarotene) is a secondary carotenoid from the same family as βcarotene, canthaxanthin, zeaxanthin, lycopene and lutein (Lorenz and Cysewski 2000). The carotenoids, astaxanthin is the highest value carotenoid since its potent antioxidative activity and more effective in scavenging free radicals (Dragoş et al. 2010). Its superior antioxidant property possesses a broad range of applications in food supplements, nutraceutical, and pharmaceutical industries as well as act as pigmentation sources for fish aquaculture (Guerin et al. 2003; Ambati et al. 2014; Fraser and Bramley 2004. Astaxanthin biosynthesis has been observed in a limited number of organisms, including bacteria, yeast Phaffia rhodozyma, fungi and in some green microalgae (Orosa et al. 2001). Among the carotenoid producing organisms, green mi croalgae are a potential resource of highvalue metabolites with the potential of producing astaxanthin (Liu et al. 2014).
However, the low productivity of these products in the native microalgae requires to be overcome (Clarens et al. 2010). Microalgae with improved growth rate and enhanced carotenoid accumulation will generate the commercial production of astaxanthin more feasible. Presently, green microalgae that have potential in accumu lating astaxanthin has received tremendous attention be cause of its high cost and the possibility of health benefits (Nakano et al. 1995). Numerous attempts have been made to improve strain with a high yield of astaxanthin. To ob tain the production process more feasible, optimization of cultivation and genetically modified strains have been ap plied for the past few decades but yet have not been fully satisfied (Kilian et al. 2011). As an alternative method, chemicals as enhancers have been proposed to initiate the production and accumulation of astaxanthin. The applica tion of chemical enhancers could be a valuable approach in addressing the low productivity of astaxanthin (Asada 1994).
Environmental oxidative stresses can enhance the massive accumulation of astaxanthin by green microal gae under the condition of high illumination, nitrogen starvation, salt stress or temperature stress (Lee and Soh 1991). The effects of these unfavorable conditions have been attributed to the formation of reactive oxygen species (ROS). The excessive ROS may damage the ability of the cells to detoxify the reactive intermediates and leading to oxidative stress conditions. These highly reactive ROS can react with lipid membranes, proteins and nucleic acids, and ultimately cause oxidative damage resulting in cell death (Lafarga et al. 2020). Therefore, ROS will be used as signal molecules in microalgae to trigger the accumula tion of astaxanthin and protect an oxidative stress damage (Apel and Hirt 2004). It might function as an effective an tioxidant with a primary line of defense against oxidative damage in scavenging free radicals (Hu et al. 2018).
Biosynthesis of astaxanthin in the cell of microal gae can be enhanced by the addition of ROS reagent (Kobayashi 2003). A study from Li et al. (2010) shown that the tolerance to excessive ROS is higher in astaxanthinrich cells with the capacity to detoxify super oxide anion radical. In addition of the iron into the culture medium, the excess levels of ROS in Haematococcus plu vialis increased ROS level. Thereby, the synthesis of fatty acids and astaxanthin was observed in the cell for protect ing their lipid vesicles (Hong et al. 2015). Induction of oxidative stress using ROS reagent in Chlorella zofingen sis, was also effective to increase the carotenoid accumu lation (Hu et al. 2018). It is known that the addition of various ROS reagents into the culture medium was able to improve the carotenoid synthesis in microalgae (Chokshi et al. 2017).
Previously, ROS generating the methyl viologen (MV) and iron ion (Fe 2+ ) compounds can lead to the formation of the superoxide anion radical and hydroxyl radical, respec tively (Kobayashi et al. 1997). The most common source of ROS reagents for astaxanthin synthesis was hydrogen peroxide (H 2 O 2 ), methylene blue (MB), and methyl vio logen (MV) for hydroxyl radicals, singlet oxygen, and su peroxide anion radicals, respectively (Ma and Chen 2001). The appropriate concentration of ROS reagent was impor tant to enhance astaxanthin formation in microalgae. Hy drogen peroxide (H 2 O 2 ) was used by Ip and Chen (2005) to generate hydroxyl radical for astaxanthin production in the heterotrophic culture of C. zofingiensis. The produc tion of astaxanthin was increased by the addition of 0.1 mM H 2 O 2 due to the formation of hydroxyl radicals (Ip and Chen 2005). The H 2 O 2 (0.1 mM) and MV (0.01 mM) was found to be the best ROS reagent in inducing caroteno genesis in Chlorococcum sp. where astaxanthin content increased almost 80% (Ma and Chen 2001). Similar re sults have been reported in H. pluvialis, the superoxide an ion radical generated from MV is the most effective ROS reagent involved in astaxanthin accumulation (Kobayashi et al. 1993).
Currently, a green microalgae species of Coelastrum sp. has proved to be a potential producer of astaxanthin. Tharek et al. (2020a) identified that Coelastrum sp. as a viable strain with the capability in producing astaxanthin from a natural source under high light intensity and nitro gen starvation in mixotrophic culture. Besides that, studies on Coelastrum sp. HA1 showed that nitrogen limitation in the culture medium of this species enhances the produc tion of astaxanthin (Liu et al. 2013). Also, Coelastrum cf. pseudomicroporum culture in municipal wastewater and salinity stress can increase carotenoid production (Úbeda et al. 2017). However, further research is required to im prove the astaxanthin content in this species for commer cial astaxanthin production. Therefore, the present study aimed to enhance astaxanthin yield using reactive oxygen species (ROS) reagent by investigating the effect of ox idative stress generated by ROS reagent of methyl violo gen towards growth and astaxanthin synthesis in tropical green microalgae strain isolated from the environment in Malaysia, Coelastrum sp. The selection strategy was fo cused on driving Coelastrum sp. into a high yield and cost effective production of astaxanthin.  (NIES collection), Japan. The cultures were grown under nor mal conditions at 25±1°C with continuous aeration and enriched with 1% CO 2 . It was illuminated at a continu ous light intensity with fluorescence light at normal pho ton flux densities (PFD) of 70 μmol photons m 2 s 1 until Coelastrum sp. cultures reach exponential growth phase for a period of 5 d. Cell growth was observed by measur ing absorbance at 750 nm using a spectrophotometer.

Supplementation culture for stress inductionn
In order to induce the astaxanthin biosynthesis, the biomass of Coelastrum sp. was harvested and various supplements were added according to optimize conditions in accumulating astaxanthin in Coelastrum sp. with de tails described in our previous work (Tharek et al. 2020b). Sodium acetate, sodium chloride and sodium nitrate were used at a final concentration of 0.5 g/L, 3 g/L and 0.1 g/L, respectively. Coelastrum sp. then was subsequently ex posed under continuous illumination of high photon flux densities (PFDs) of 250 μmol photon m 2 s 1 . The cells were then subjected to extraction of astaxanthin and all the experiments were carried out in triplicates.

Exposure of reactive oxygen species generating reagent
Methyl viologen (MV) is a reactive oxygen species reagent that can produce superoxide anion radical (O 2 ) (Rabinow itch et al. 1987

Determination of chlorophyll
Microalgae culture at the end of the exponential phase (5 day culture) was subjected for analysis. The 200 µL of mi croalgae culture was treated with 80% acetone. The mix ture was vortexed for 30 s and centrifuged at maximum speed for 5 min. The absorbance of extracted chlorophyll was read at 663.6 nm and 646.6 nm. The concentration of chlorophyll a, b and total chlorophyll were calculated by the Lichtenthaler equation and expressed in mg/L content (Lichtenthaler 1987).

Determination of astaxanthin
To measure the astaxanthin content, a known volume of microalgae culture was taken and centrifuged at 2000 × g for 10 min. The pellet was then lyophilized using a freeze dryer (Lyphlock 6; Labconco, USA). Then, the carotenoids were extracted using solvent extraction by ho mogenized the cells with acetone and kept in a water bath at 70°C for 10 min followed by vortexing for few min utes. The mixture was centrifuged at 2000 × g for 10 min and the supernatant was collected. Supernatant collections were conducted repeatedly until the cells were faded. The astaxanthin concentration was then measured by the spec trophotometric method and calculated with the equation, c (mg/L) = 4.5 × A 480 × (V a / V b ) × f. Where c is the as taxanthin concentration, V a (mL) is the volume of solvent, V b (mL) is the volume of algal sample, and f is the dilution ratio. The absorption peak of astaxanthin is at 480 nm and thus, A 480 was determined by measuring the absorbance at 480 nm. Acetone was used as blank for the measurement.

Statistical analysis
The experiment was carried out with replication from three separate cultures. All values shown in the figures are ex pressed as mean SD. Student's t test was used to determine significant differences.

Results and Discussion
In general, there are two crucial roles of carotenoids in photosynthetic organisms. First, they act as light harvesting pigments by trapping light energy and pass ing it to chlorophylls. Second, and more importantly, carotenoids can quench singlet oxygen (1O 2 ) by protect ing the photosynthetic apparatus from unfavorable condi tions (Young 1991). Shaish et al. (1993), have reported that massive amount of carotenoid accumulated in green algae cells are involved in triggering ßcarotene biosyn thesis to protect the photosynthetic cell against oxidative stress. Under unfavorable conditions, such as high light, salt stress or nutrient deprivation, the reactive oxygen species (ROS) was generated in the chloroplast when the photosynthetic process and CO 2 fixation were perturbed (Mittler 2002). The ROS will be produced whenever there is excessive reducing power in photosynthesis and will then be used as signal molecules to initiate the production and accumulation of many bioproducts (Asada 1994).
Only few studies have focused on the involvement of oxidative stress using ROS reagent in carotenoid synthesis (Lafarga et al. 2020). In the present study, the addition of  . All data represent an average of 3 replications and error bars indicate mean ±SD. Statistical analyses were conducted using student's t-test. Different small letter represents significant different among control and different treatments. Small letters a and b above the bar graph indicates significant increases and decreases respectively, between control and treatments groups (P<0.05). *a for groups that higher than control; and b for groups that lower than control reactive oxygen species (ROS) reagent, which was methyl viologen (MV), can rapidly auto oxidizes to produce su peroxide anion radical (O 2 ). To investigate the tolerance of Coelastrum sp. cells towards ROS reagent, different concentrations of MV were tested by growing 10% inocu lums of Coelastrum sp. in the presence of MV and shaken manually daily. Based on the results obtained, the growth of cultures was markedly decreased after the 4th day in 0.01 mM, 0.1 mM and 1.0 mM of MV, as shown in Fig  ure 1. Besides, the microalgae cultures in these conditions turn to white depicting the death of cells. The growth of culture in 0.0001 and 0.001 mM of MV was shown to in crease even after the 4th day of incubation, indicating the ability of the cells to survive in this range of MV concen tration.
To investigate further the effect of superoxide anion radical (O 2 ) towards astaxanthin synthesis, ROS reagent (MV) was added to Coelastrum sp. culture during the ex ponential growth phase (5day culture) where a rapid uti lization of the substrate and cell division occurred at this stage. The same cell density was applied for all cultures supplemented with MV. The parameters included in the study to examine the effect of MV were the growth of Coelastrum sp., Chlorophyll content and astaxanthin con tent. Figure 2 shows the growth of Coelastrum sp. grown after incubated under different concentrations of MV en riched with 1% of CO 2 enrichment.
The growth of Coelastrum sp. without the addition of MV (Control) was observed to be higher compared to cul tures supplemented with MV. In contrast, at higher con centration (0.1 mM and 1.0 mM), the growth of Coelas trum sp. were significantly decreased; therefore, the ac cumulation of astaxanthin was inhibited (Figure 3). The At the beginning of culture (day 0), the algal cells were in the green color relatively because of high chlorophyll content and low carotenoid content. With the addition of MV, the astaxanthin production proceeded markedly with a reduction of chlorophyll content, as shown in Figures  3 and 4. Superoxide anion radical generates by MV was found to be more effective for astaxanthin production at an extremely low concentration of 0.001 mM and 0.0001 mM. The results obtained in Figure 3 showed that MV at 0.001 mM increased astaxanthin content with 1.3 times higher than control after seventh day of incubation depict ing the highest astaxanthin content. While the color of Coelastrum sp. changes to orangish color after 7 d of incu bation under the lower concentration of MV as depicted in Figure 5 indicating the faster accumulation of carotenoids. However, the production of astaxanthin did not proceed at 0.01 mM MV and the astaxanthin content was found to decreased about 50% after 7 d of incubation, as shown in Figure 3 suggesting the low astaxanthin accumulation may be due to the free radicals being scavenged (Raman and Ravi 2011). At high concentration of 0.1 mM and 1.0 mM MV, the growth of microalgae was reduced and inhibited astaxanthin accumulation.
In corroboration of this findings showed that astaxan thin rich cells are more effective to the concentration that can tolerate with the cells of microalgae. Methyl violo gen, which generated superoxide anion radical (O 2 ), was capable to trigger the astaxanthin synthesis in Coelastrum sp. and effective at low concentration of MV. This radi cal might enhance carotenoid formation in microalgae cyst cells by participating directly in the carotenogenic enzyme reactions as an oxidizer (Kobayashi et al. 1993). There fore, the accumulation of carotenoid acts as a protective agent against oxidative stress damage (Shaish et al. 1993).
However, excessive addition of MV could cause massive cell death in the end and drastically reduced astaxanthin formation. Astaxanthin plays a vital role in protecting the algal cells against oxidative damage of reactive oxygen species. Consequently, the cell of microalgae has devel oped an efficient defense system to helps it to survive un der unfavorable condition.

Conclusions
To produce bioproducts in an economically feasible way, the low productivity of microalgae needs to be addressed. Therefore, methyl viologen as reactive oxygen species (ROS) reagent has been applied as an enhancer to improve the accumulation of high yield of astaxanthin from Coelas trum sp. In this study, we concluded that the methyl violo gen reacts as ROS reagent by generating superoxide anion radical at low concentration of MV (0.001 mM) and con sequently lead to highest astaxanthin production with 1.3 times higher than control.