Enhanced production of astaxanthin at different physico-chemical parameters in the green alga Haematococcus pluvialis Flotow

Enhanced production of astaxanthin at different physico-chemical parameters in the green alga Haematococcus pluvialis Flotow S. Nagaraj a*, P. Arulmurugan a, M. G. Rajaram a, R. Sundararaj b and R. Rengasamy a a Centre for Advanced Studies in Botany, University of Madras, Guindy Campus, Chennai, India b Govt. Arts College, Nandanam, Chennai, India *Corresponding author: Email: nagalilly@gmail.com Abstract In the present study, the effects of ph, light intensities, various inorganic nitrogen sources, NaCl, K 2HPO 4 and different light periods were investigated on the vegetative cells of Haematococcus pluvialis Flotow. Maximum level of total carotenoids were (68.59 µg/ml) at ph 7.0, light intensities (73.57 µg/ml) at 40.0 µem -2 s -1, 0.2 M of NaNO 3 (67.49 µg/ml), 2.0 mm of CH 4N 2O (63.32 µg/ml) and 2.0 mm of NH 4Cl (66.21 µg/ml), 0.06 M of K 2HPO 4 (77.65 µg/ml) 0.06 M of NaCl (68.26 µg/ml) correspondence to maximum number of cells 6.62, 6.31, 6.31, 6.52, 6.81, 6.51 and 6.41 log 10 cell number/ml respectively. In addition, the alga kept under different light periods, the organism had maximum production of carotenoid of 69.50, 98.65 and 37.55 µg/ml under mixotrophic, phototrophic and heterotrophic conditions, respectively. Based on the investigation a Modified HP medium-i was proposed and compared the growth characteristics of the test alga with Modified 3N+BBM+V medium. The organism had maximum content of carotenoids of 97.13 µg/ml at Modified HP medium-i as against 78.32 µg/ml with Modified 3N+BBM+V medium on 27 th day. It was concluded that phosphate and light intensity were greatly influencing the accumulation of total carotenoids of the alga. Keywords: Carotenoids, Haematococcus pluvialis, Light intensities, Nitrogen Introduction The "green algae" comprise of more than 7000 species growing in a variety of habitats. Development of commercial-scale microalgal culturing techniques is being carried out globally for the production of bioactive compounds, aquaculture feed, fine chemicals, and renewable fuels. Haematococcus pluvialis is a eukaryotic, unicellular, motile, biflagellate, green, fresh water alga capable to grow under both photoautotrophic and heterotrophic conditions (Sarada et al., 2002a, Kang et al., 2005). Under favorable growth conditions, it exists as a single biflagellate swimmer capable of showing autotrophic growth. During unfavorable growth conditions, H. pluvialis initiates carotenogenesis and undergoes morphological transformation from green vegetative cells to deep red, astaxanthin-rich, immotile aplanospores (Harker et al., 1996a). Thus a distinct two morphological phases viz., green motile vegetative phase and red non-motile carotenoid accumulated encysted (aplanospore) phase exist in the life history of H. pluvialis. The conditions such as nutrient limitations, oxidative stress, elevated temperature, intense light intensity and salinity represent the unfavorable growth conditions, also referred as stress factors or inductive conditions (Kobayashi et al., 1993; Tjahjono et al., 1994; Sarada et al., 2002b; Jin et al., 2006). During the morphological transformation, a trilaminar sheath and acetolysis resistant material are formed and thickened, coinciding with massive accumulation of astaxanthin (Jhonson and Schroeder, 1995; Monstant et al., 2001). Astaxanthin enables Haematococcus to acclimate to high light by dissipating excessive light energy and shielding photosynthetic apparatus (Wang et al., 2003). Subsequently, after being exposed to favorable environment, cysts revert to the mobile phase. The carotenoid pigment astaxanthin (3, 3 - dihydroxy-β, β-carotene-4, 4 -dione) has important applications in nutraceutical, cosmetics, food and feed industries and as a potent antioxidant of its strong antioxidant activity to target several health conditions, approximately 10 times greater than other carotenoids, such as zeaxanthin, lutein, canthaxanthin, and β-carotene, and over 500 times greater than α-tocopherol (Tripathi et al., 1999). The present investigation was aimed to optimize culture conditions to enhance growth and carotenoid production in Haematococcus pluvialis Flotow under laboratory conditions. Materials and Methods Haematococcus pluvialis Flotow The green algal culture of Haematococcus pluvialis Flotow was obtained from the Central Leather Research Institute, Chennai (they received from the Culture Collection of the University of Göttingen, Germany). It was maintained in Modified 3N-BBM+V medium at 25 1 o C, 30µEm -2 s -1 light irradiance and photoperiod of 12/12 (Light/Dark). The cultures were mixed manually twice a

day. Axenic culture of the alga was obtained after antibiotic treatment (Droop, 1967) used in the following experiments. Optimization parameters and growth conditions for total carotenoids synthesis Haematococcus pluvialis grown at different initial ph: 5.0, 5.5, 6.0, 6.5 (control), 7.0, 7.5 and 8.0; light intensities: 2, 4, 10, 20, 30 (control) and 40 µem -2 s -1 ; different concentrations of NaNO 3 : 0.1, 0.2, 0.3 (control), 0.4, and 0.5M; CH 4 N 2 O: 1.0, 2.0, 3.0, 4.0 and 5.0Mm; NH 4 Cl: 1.0, 2.0, 3.0, 4.0 and 5.0mM; K 2 HPO 4. 3H 2 O: 0.02, 0.04 (control), 0.06, 0.08 and 0.1M; NaCl: 0.02, 0.04 (control), 0.06, 0.08 and 0.1M. Further, the alga, grown kept under different light periods: 12/12 Light/Dark, continuous light and continuous dark and recorded different parameters. Ten ml of optimally grown cultures of H. pluvialis was inoculated in 90 ml of Basal medium and kept under the laboratory conditions. This experiment was conducted for a period of 30 days. At every three days interval the following parameters: i) Cell number (Neubauer haemocytometer) (Cn) and ii) concentrations of pigments viz., Chlorophyll a (Chl a), Chlorophyll b (Chl b) and Total carotenoids (Tc) (µg/ml) (Lichtenthaler, 1987) were recorded. In addition, growth curves were plotted against days and log 10 of cell number. All the experiments were carried out in triplicates and the mean values are represented and discussed. Results Haematococcus pluvialis culture conditions The different stages of green alga, Haematococcus pluvialis Flotow grown under the laboratory conditions Figure 1. Figure1. Different stages of Haematococcus pluvialis Flotow under laboratory condition Optimization of conditions for enhanced total carotenoids content in Haematococcus pluvialis Effect of different initial ph Haematococcus pluvialis survived in all the different initial ph chosen in the basal medium. The isolate showed a gradual increase in cell number from ph 5.0 to 7.0. Maximum growth of 6.621 log 10 of cell number/ml was recorded at ph 7.0 on 18 th day. The increment in cell number at this condition was more than 5.0% to that of control (ph 6.5) (Fig. 2). Further, H. pluvialis grown at ph 7.0 showed maximum concentrations of Chl a and Chl b of 7.053 µg/ml and 3.71 µg/ml, on 21 st and 18 th day, respectively. In addition, the organism at ph 7.0 registered a maximum total carotenoids content of 68.51 µg/ml on 27 th day. Hence the ph 7.0 was included in the following experiments (Figs. 3, 4, 5).

Effect of different light intensities On 18 th day, the organism showed a maximum growth by 6.319 log 10 of cell number/ml at 40 µem -2 s -1 on 18 th day. (Fig. 6). At this condition the organism contained maximum concentration of Chl a 7.41 µg/ml and Chl b 4.75 µg/ml on 15 th day. The increment of Chl b was more than 20% to that of control (30 µem -2 s -1 ). Further maximum amount of 73.57 µg/ml of total carotenoids recorded on 27 th day was more than 15% (Figs. 7, 8, 9). Among the different light intensities chosen the organism exhibited good growth and maximum accumulation of pigments at 40 µem -2 s -1. Therefore this condition was included in the following experiments.

Effect of different concentrations of NaNO 3 The alga Haematococcus pluvialis showed growth in the medium amended with different concentrations of NaNO 3. Maximum cell number of 6.314 log 10 /ml was recorded 0.5M NaNO 3 on 21 st day. The increment at this condition was more than 10% to that of control (0.3M) (Fig. 10). Similarly maximum concentrations of Chl a and Chl b of 7.832 µg/ml and 5.421 µg/ml recorded on 21 st and 18 th days were more than. 35% and 15%, respectively, to that of control. The organism accumulated maximum amount of total carotenoids of 67.49 µg/ml at 0.2M NaNO 3 on 27 th day, which was more than 15% to that of control (Figs. 11, 12, 13). Among the different NaNO 3 concentration tested amendment of 0.2M NaNO 3 favored the organism for maximum accumulation of total carotenoids. Therefore, this concentrations was included in the following experiments Effect of different concentrations of CH 4 N 2 O (Urea) The green alga H. pluvialis survived in the medium amended with different concentrations of urea. The cell number of the organism increased steadily up to 21 st day. On this day it reached a maximum of 6.521 log 10 of cell number/ml at 2.0 mm CH 4 N 2 O and it was similar to control (3N+BBM+V medium) (Fig. 14). Haematococcus pluvialis contained a maximum Chl a content of. 6.42µg/mL at 5.0 mm on 21 st day while Chl b recorded a maximum of 3.73 µg/ml at 2.0mM urea on 18 th day. The amount of total carotenoids of the isolate was registered a maximum value of 65.32 µg/ml on 27 th day at 2.0mM CH 4 N 2 O (Figs. 15, 16, 17). Amendment of urea in the medium did not much enhance the growth and pigment content of the organism. Therefore this was not included in the following experiments

Effect of different concentrations of NH 4 Cl Haematococcus pluvialis survived in the medium amended with different concentrations of NH 4 Cl chosen. The organism grew well of 2.0mM, 3.0mM, 4.0 mm, and 5.0 mm NH 4 Cl amendment. The algal cell number was increased steadily up to 21 st day. On this day, a maximum of 6.814 log 10 cell number/ml was recorded at 2.0mM NH 4 Cl as similar to control (Fig. 18). Maximum concentrations of Chl a and Chl b of 6.92 µg/ml and 5.45 µg/ml were recorded at 5.0 mm and 4.0mM NH 4 Cl on 21 st day and 18 th day, respectively. On 30 th day, H. pluvialis grown at 2.0mM NH 4 Cl showed maximum total carotenoids content of 64.12 µg/ml (Figs. 19, 20, 21). Growth by cell number and concentrations of different pigments did not show much increment to that of control. Therefore this chemical was also not included the following experiments.

Effect of different concentrations of K 2 HPO 4 The green alga, H. pluvialis grew well in the medium amended with different concentrations of K 2 HPO 4. The number of cells increased steadily up to 21 st day. On this day a maximum number of cells of 6.51 log 10 /ml was recorded at 0.1M K 2 HPO 4. The increment in cell number was more than 17.0% when compared to control (0.04M) (Fig. 22). Maximum concentrations of Chl a and Chl b of 8.32 µg/ml and 4.9 µg/ml were recorded in H. pluvialis at 0.1M K 2 HPO 4 and 0.08M K 2 HPO 4 on 21 st and 18 th day, respectively. The increment of Chl b was more than 30.0% to that of control. A maximum total carotenoids content of 77.65µg/mL recorded at 0.02M KH 2 PO 4 on 27 th day, was more than 20% when compared to control (Figs. 23, 24, 25). Therefore, 0.02M K 2 HPO 4. 3H 2 O was included in the following experiments. Effect of different concentrations of NaCl Haematococcus pluvialis survived in the medium amended with different concentrations of NaCl chosen. The algal cell number was increased steadily up to 21 st day. On 21 st day, a maximum of 6.419 log 10 cell number/ml was registered at 0.06M NaCl. The increment in the cell number was more than 30%, when compared to control (0.04M) (Fig.26). Similarly, maximum concentrations of Chl a and Chl b of 6.613 µg/ml of 4.52 µg/ml were recorded at 0.06M NaCl on 21 st day and 18 th day respectively. The increment of Chl a was more than 24% to that of control.

However on 30 th day, H. pluvialis grown at 0.06M NaCl contained a maximum amount of total carotenoids of 68.26 µg/ml, which was more than 15% when compared to control (0.04M) (Figs. 27, 28, 29). Therefore, in the following experiments 0.06M NaCl was included in the basal medium. A modified HP medium-i was proposed based on the above investigation. Comparative study of H. pluvialis grown in Modified 3N+BBM+V medium and HP medium I The green alga, Haematococcus pluvialis showed maximum growth in the HP medium-i than Modified 3N+BBM+V medium (Fig. 30b). Maximum cell number of 7.13 log 10 /ml recorded in the former medium was more than 10.0% to that of control on 18 th day (Fig. 30a). Similarly, maximum amounts of Chl a and Chl b of 8.3 µg/ml and 2.9µg/mL recorded in H. pluvialis on 18 th and 15 th day were more than 15% and 7%, respectively. (Fig. 30a). Maximum total carotenoids content of 97.13µg/mL was recorded in the formulated HP medium-i as against 78.32 µg/ml in Modified 3N+BBM+V medium on 27 th day (Fig. 30b).

Effect of different light conditions The unicellular green alga, Haematococcus pluvialis grew in the HP medium-i under three different light periods: 12/12 light/dark, continuous light and continuous dark conditions revealed that the organism had maximum levels of Chl a and Chl b of 5.4 µg/ml and 2.9 µg/ml under continuous light condition on 18 th and 15 th day, respectively. In addition, maximum total carotenoids and astaxanthin levels of 93.3µg/mL and 61.2µg/mL were recorded on 30 th day. (Figs. 31, 32 and 33). Discussion An attempt was made in the present study to enhance the production of carotenoid in the isolate of H. pluvialis since it accumulates high amount of carotenoids. Two major aspects are usually considered for improvement in developing an optimal process for microalgal products. The first aspect is the optimization of environmental factors such as temperature, light intensity and ph and the second is the selection of a suitable nutrient medium. It is well known that the culture medium not only affects the cell productivity, but also affects the cell composition and yield of specific products (Shay et al., 1987, Gong and Chen, 1997).

Astaxanthin from Haematococcus was first postulated to be a storage material and its accumulation being supported by the deficiency in nitrogen, phosphorous or any other nutrient that retards cell multiplication and as long as carbon is available (Pringsheim 1966). Astaxanthin is expected to accumulate when the cell divisions are ceased or expected to accumulate while the cells are growing actively, in order to protect the photosynthetic apparatus. Astaxanthin accumulation was more pronounced during aplanospore stage, while the cells ceased to divide (Borowitzka et al. 1991; Fábregas et al., 2001). Higher light intensities can lead to photoinhibition. Light penetration (which is inversely proportional to cell concentration) is another problem in the phototrophic cultivation of microalgae (Margalith, 1999). The effect of light intensity is dependent on the nutritional state of the cultures. Haematococcus pluvialis accumulates large amounts of ketocarotenoid astaxanthin in response to high light irradiation, nitrogen limitation and salt stress (Yong and Lee, 1991; Kobayashi et al., 1992; Boussiba, 2000). Among the different light intensities chosen in the present study, the alga exhibited maximum cell number of 6.319 log 10 /ml on 18 th day and the concentrations of Chl a of 7.41 µg/ml and Chl b 4.52 µg/ml on 15 th day at the high light intensity of 40 µem -2 s -1 chosen. Throughout the study period the accumulation of total carotenoids of the alga was increased from 21 st day, whereas, the levels of Chl a and Chl b were found decreased. On 27 th day the present alga, H. pluvialis contained 36.58 pg/cell of carotenoids whereas, Cifuentes et al. (2003) recorded only 25.0 pg/cell - 1. Kim et al. (2006) recorded maximum carotenoids of 99 µg/ml under high light intensity. Carotenoid accumulation in H. pluvialis coincided with a decline in the photosynthetic activity (Boussiba and Vonshak 1991). In the present study also carotenoid accumulation was increased in the later period of growth and in contrast the levels of photosynthetic pigments such as Chl a and Chl b were decreased. High light intensity over a period of time induced total carotenoids synthesis. It revealed that a change in the amount of light per amount of cell would influence the synthesis of carotenoids (Hejazi and Wijffels, 2003). The effect of high light irradiance was undoubtedly the most important factor in the astaxanthin accumulation as reported by Harker et al. (1996), Lee (1999), Boussiba (2000), Park and Lee (2000, 2001), Steinbrenner and Linden (2003) and Chio et al. (2003). ph is one of major factors to surpass cell growth and maximum production of carotenoids (Lee and Zhang, 1999). Among the different environmental stresses, ph has been the greatest effect on the morphological changes on H. pluvialis cells and carotenoid formation. It determines the solubility of carbon dioxide and minerals in the culture and directly or indirectly influences the metabolism of the organisms. In the present attempt, H. pluvialis was survived in all the different initial ph chosen in the basal medium viz. 5.0, 5.5, 6.0, 6.5 (control), 7.0, 7.5 and 8.0. The alga showed a gradual increase in cell number at ph ranging from 5.0 to 7.0. Maximum growth by 6.621 log 10 of cell number/ml was recorded at ph 7.0 on 18 th day, which was more than 5.0% to that of control (ph 6.5). Similarly, the organism grown at ph 7.0 had maximum concentrations of Chl a and Chl b of 7.053 µg/ml and 3.71µg/mL, on 21 st and 18 th day, respectively, and the total carotenoids up to 68.51µg/mL (17.0 pg/cell) on 27 th day. The organism kept in alkaline conditions showed poor growth and less accumulation of total carotenoids. Exposure of H. pluvialis to a nitrogen-deprived medium has been an effective condition for enhancing astaxanthin accumulation (Zlotnik et al., 1993; Harker et al., 1995, 1996; Hagen et al., 2000). Nitrogen deficiency as one of the crucial factors blocking cell division and stimulating the synthesis of astaxanthin (Borowitzka et al., 1991). Among the three nitrogen sources chosen in the present study such as, NaNO 3, Urea and NH 4 Cl the alga preferred NaNO 3 for its maximum growth and total carotenoids accumulation. The growth in terms of cell numbers was maximum at the high concentration of NaNO 3 at 0.5M. At this condition the Chl a content was increased up to 2 folds to that of control. However, the accumulation of total carotenoids was maximum at the low concentration of nitrogen, NaNO 3 at 0.2M up to 67.49 µg/ml, (33.57 pg/cell -1 ) on 27 th day. Our results are in accordance with the observations made on H. pluvialis by Borowitzka et al. (1991) and Cifuentes et al. (2003). Cifuentes et al. (2003) recorded only 25.2 pg/cell -1 of carotenoids in H. pluvialis on 12 th day. Fábregas et al. (1998) reported that N- deficiency and high light intensity increased the astaxanthin accumulation up to 49.52 µg/ml. High levels of nitrogen in the medium place a demand on carbon skeletons or photosynthates for assimilation and the reduced nitrogen could result in a competition for carbon between carotenoids synthesis and aminoacid synthesis. This may be the reason for reduced levels of carotenoids at high levels of nitrogen in the medium and for the accumulation of huge quantities of carotenoids under nitrogen deprived conditions (Borowitzka et al., 1991). Thus, nitrogen deficiency seems to be the most important factor triggering the synthesis of carotenoids.

Under favorable environmental conditions, H. pluvialis is at green vegetative condition and it produces less amount of astaxanthin. Astaxanthin accumulation can be induced in H. pluvialis during the transformation of vegetative condition to the aplanospore stage as a response to various stress-inducing conditions such as nitrogen limitation, strong light intensity, salt stress, phosphate deficiency (Harker et al. 1996; Fábregas et al. 1998, 2001; Margalith 1999; Hata et al. 2001; Sarada et al. 2002a; Choi et al. 2003; Wang and Zarka 2003). The accumulation of astaxanthin in cysts under salt stress conditions has been reported in H. pluvialis (Borowitzka et al., 1991; Boussiba and Vonshak, 1991; Boussiba et al., 1992; Cordero et al., 1996; Kobayashi et al., 1997; Harker et al., 1995, 1996 a, b). Mortality of H. pluvialis cells increasing substantially with the increasing salt concentration and only 55% of cells survived at 0.8% NaCl (138 mm) (Harker et al. 1996a, b). In spite of this high mortality, an increase in red coloration of the surviving cells at higher salinity was reported. Sarada et al. (2002) stated that the age of culture was crucial to trigger astaxanthin production in salt stress induced culture. Haematococcus pluvialis required a longer inductive period for the accumulation of high carotenoids content (Sarada et al., 2002). However the level of carotenoids accumulated by salt stressed cells was low as observed by Harker et al. (1995, 1996a). In the present attempt, the algal cell number increased steadily up to 21 st day and a maximum of 6.419 log 10 cell number/ml was registered at 0.06M NaCl. The increment in the cell number was more than 30% when compared to control (0.04M). Similarly, 0.06M NaCl favoured maximum concentration of Chl a which was more than 24% to that of control. On 30 th day, H. pluvialis grown at 0.06M NaCl had a maximum total carotenoids amount of 27.07 pg/cell -1, as similar to the observations made by Harker et al. (1996b). Harker et al. (1995), studied the effectiveness of addition of NaCl on the carotenogenesis of H. pluvialis strain CCAP 34/7. They observed that improved astaxanthin yield was possible when the organism grown in lower amount of NaCl and expose to very high PFD (1600-1700 µmol m -2 s -1 ). In this attempt, it was observed that the concentrations above 0.06M NaCl decreased the accumulation of astaxanthin in H. pluvialis. Phosphorus is an another important nutrient for algal growth. It is responsible for the energy transfer of cells and the formation of cell membranes and nucleic acids. Besides being a structural element in nucleic acid and phospholipids it plays crucial roles in various biological functions such as energy transformations, activation of metabolic intermediates, signal transduction cascades and regulation of enzymes. It also plays an important role in cell energetic through high-energy phosphodiester bonds (ATP, sugar-phosphates) and in intracellular signaling (phosphorylation and dephosphorylation of proteins) (Geider et al., 1998). Moreover, phosphate plays significant roles in the syntheses of valuable products such as astaxanthin and PUFAs. Brinda et al. (2004) reported that high biomass of 3.5 g l -1 and astaxanthin 15.0 mg g -1 was achieved in H. pluvialis under in the PO 4 -deficient condition. In another green alga, Chlorococcum sp., the cell growth was saturated at a relatively high PO 4 level while the secondary carotenoids were preferably accumulated at a relatively low PO 4 level. Low concentration of K 2 HPO 4 favored H. pluvialis for maximum production of pigments (Kang et al., 2006). In the present study, the medium amended with 0.1M and 0.08M of K 2 HPO 4 favored the organism for the synthesis of maximum concentrations of Chl a and Chl b of 8.32 µg/ml and 4.9 µg/ml on 21 st and 18 th day, respectively. Whereas low concentration of KH 2 PO 4 (0.02M) accelerated maximum synthesis of total carotenoids up to 24.3 pg/cell -1 in H. pluvialis on 27 th day. Ping et al. (2007) had recorded only 11.0 pg/cell -1 carotenoids content in phosphate deficiency condition. Boussiba and Vonshak, (1991); Boussiba et al., (1992) demonstrated that it was possible to induce astaxanthin accumulation in the cultures of H. pluvialis by depletion of phosphate in the growth medium or by increasing light intensity. The amount of total carotenoids was enhanced under continuous light (phototrophic) conditions under all effective culture ratios when compared to low light intensity. This indicated that carotenoids biosynthesis is photo dependence (Kobayashi et al., 1992a). They also reported that light quantity defined as the multiplication of light intensity by the net illumination time. It is an important parameter in carotenoids biosynthesis than light intensity alone. Light quantity rather than light intensity has been correlated with carotenoid content in H. pluvialis, with continuous light being more effective than light-dark cycles (Kobayashi et al. 1992b). In the present study among the three different experiments conducted i) continuous light (phototrophic), light/dark (mixotrophic) and continuous dark (heterotrophic) on the organism, H. pluvialis, and it preferred phototrophic condition for maximum astaxanthin production up to 64.10µg/mL. In general, after growth phase of Haematococcus cells tend to settle at the bottom of culture flasks and undergo

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