Effect of ph on growth and biochemical responses of Dunaliella bardawil and Chlorella ellipsoidea

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1 World J Microbiol Biotechnol (21) 26: DOI 1.17/s z ORIGINAL PAPER Effect of on growth and biochemical responses of Dunaliella bardawil and Chlorella ellipsoidea Zeinab I. Khalil Mohsen M. S. Asker Salwa El-Sayed Imam A. Kobbia Received: 21 October 29 / Accepted: 15 December 29 / Published online: 3 December 29 Ó Springer Science+Business Media B.V. 29 Abstract The goal of the present investigation was to study the effect of on growth and biochemical responses of Dunaliella bardawil and Chlorella ellipsoidea when exposed to different values. The two tested microalgae could grow in a wide range of (4 9 for D. bardawil and 4 1 for C. ellipsoidea). The dry weight gain and the biochemical components of D. bardawil were greatly enhanced at 7.5. In contrast, dry weight and carbohydrate content of C. ellipsoidea attained their maximum values at the alkaline. On the other hand, the protein content of C. ellipsoidea recorded its highest value at 4, while the pigment content of the same alga was highest at 4, 6, and 7.5 and decreased at alkaline. Both 6 and 9 stimulated the accumulation of b-carotene, vitamin E and vitamin C in D. bardawil, with the highest values of the three compounds recorded at 9. In the case of C. ellipsoidea, b-carotene content increased at 6 and 1 as compared with the control, but the amount of b-carotene was much higher at 6 than at 1. Vitamin E content was higher in C. ellipsoidea cells at 1 than at 6. Both 6 and 1 caused a significant decline in vitamin C content of C. ellipsoidea. Imam A. Kobbia Late professor of Phycology. Z. I. Khalil S. El-Sayed I. A. Kobbia Department of Botany, Faculty of Science, Cairo University, Cairo, Egypt M. M. S. Asker (&) Microbial Biotechnology Department, National Research Center, Dokki, Cairo, Egypt mohsenmsa@yahoo.com Keywords Green microalgae Dunaliella bardawil Chlorella ellipsoidea Antioxidants b-carotene Vitamin E Vitamin C Introduction Algae of the genus Dunaliella and Chlorella are among the most studied microalgae for mass culture. They are grown as a food source in aquaculture and for the production of high value protein and other bioactive constituents. More recently, these microalgae have been grown under certain conditions, e.g., nutrient stress, high light intensity, high salt concentration and extremes that induce the accumulation of highly important antioxidants (Matsukawa et al. 2; Ip and Chen 25). For example, Dunaliella can produce and accumulate large amounts of commercially important compounds such as b-carotene and glycerol in response to stress conditions. Chlorella, a virtually ubiquitous unicellular green alga, has been also used for commercial production of functional foods (Pelah et al. 24). Variations in the environmental conditions away from those which are potential result in the accumulation of potentially harmful reactive oxygen species (ROS), viz., superoxide (O 2 - ) and hydroxyl (OH ) radical, which can overcome the antioxidant defenses of the host, thus producing (often irreversible) cellular damage (Grace and Logan 1996; Giao et al. 28). ROS also include singlet oxygen ( 1 O 2 ), superoxide radical anions and hydrogen peroxide (H 2 O 2 ) (Asada 1999). These ROS attack different cell organelles, causing a series of deteriorative changes in the biological system leading to cell inactivation (Imlay and Linn 1988). Plants respond to elevated ROS levels by

2 1226 World J Microbiol Biotechnol (21) 26: activating a number of anti-oxidative defense mechanisms to combat the danger posed by the presence of ROS. ROS are scavenged enzymatically or non-enzymatically with small molecules, i.e., carotenoids, tocopherol and ascorbic acid. Thus the measurement and identification of the antioxidants, which can block the generation of ROS, has gained importance in recent years (Özyürek et al. 28). b-carotene protects the cells against oxidative damage by inhibiting or quenching free radicals and ROS (Hunter and Willer 1994). b-carotene is a lipophilic highvalue compound; it has been traditionally commercialized in food additives including colorants, antioxidants and vitamins (Mojaat et al. 28) and thus is widely used in the food, pharmaceutical and cosmetic industries. Apart from being precursor for vitamin A synthesis in the body, the antioxidant activity of b-carotene is well known. One molecule of b-carotene is found to neutralize up to 1, molecules of free radical oxygen (Dufosse et al. 25). Vitamin E comprises a group of lipophilic compounds of which a-tocopherol is the most abundant and has the highest antioxidant activity in vivo (Matsukawa et al. 2). It is well known that vitamin E is an essential micronutrient involved in various processes relevant to human health and disease. Recently, it is strongly believed that vitamin E functions in the regulation of cellular singling processes and gene expression (Frank 25). Vitamin C is important for its anti-oxidative and metabolic functions in both animals and humans (Ushimaru et al. 26): it helps the body in forming connective tissues, bone, teeth, and blood vessel walls. Its role as in vivo antioxidant has received much attention over the past decade. It is also a primary defensive vitamin through its function as a free radical scavenger, including quenching of singlet oxygen (Eitenmiller and Landen 1999). The hydrogen ion concentration () of the culture medium is one of the most important factors that seriously affect the optimal growth of algal cultures. is very important for the character of metabolism of microorganisms and hence for the biosynthesis of the bioactive products as secondary metabolites. Liu and Lee (2) indicated that affected both quantity and composition of carotenoids in Chlorococcum sp. On the other hand, the Cd and Zn removal from solution by Scenedemus obliquus and Desmodesmus pleiomorphus depends on, with a maximum level of removal at ca. 7. and 5, respectively (Monteiro et al. 29a, b). The aim of the present study was to determine the growth and biochemical responses of two unicellular chlorophycean microalgae, Dunaliella bardawil and Chlorella ellipsoidea, when exposed to different -induced oxidative stresses. The capability of microalgae to accumulate antioxidants such as b-carotene, a-tocopherol (vitamin E), and L-ascorbic acid (vitamin C) under inductive conditions was also studied. Materials and methods Microorganism and culture conditions Dunaliella bardawil was kindly supplied by Prof. Abdel- Fattah Khaleafa, Professor of Phycology, Botany Department, Alexandria University, Alexandria, Egypt, Chlorella ellipsoidea was obtained from algal collection center, Mansoura University, Mansoura, Egypt. The axenic cultures of D. bardawil and C. ellipsoidea were maintained in MH (Loeblich 1982) and MBL (Nichols 1973) nutrient media, respectively. Erlenmeyer flasks (25-ml) each containing 5 ml of the nutrient medium were prepared. Various values were adjusted to 4, 6, 7.5, 8, 9, 1, and 11 using NaOH or HCl solutions. Control and the treated flasks were inoculated with the algae. The start inoculum density was adjusted to an arbitrary standard cell number of and cells ml -1 of D. bardawil and C. ellipsoidea, respectively. All the culture flasks were incubated at 28 ± 2 C under continuous illumination (using florescent lamb) at 78 le m -2 s -1. The duration of each experiment was 1 days as determined from the growth curves of both microalgae. All the experiments were carried out in triplicate. Analytical determinations The biomass of treatments and control were separated from media by centrifugation. Three replicates from each treatment were directly put in a hot dry oven at 7 8 C for 24 h or till constant weight to determine the changes in the dry weight. Another set was extracted with 9% acetone and the color intensity was measured at 663, 647, and 452 nm for pigment estimation (Metzner et al. 1965). The total carbohydrate content was measured according to the method of Dubois et al. (1956), using glucose as a standard. The procedure of Lowry et al. (1951) was adopted for the estimation of protein in the different dry biomasses, using bovine serum albumin (BSA) as standard. Glycerol was estimated in D. bardawil cultures according to Chitlaru and Pick (1991). HPLC analysis The contents of b-carotene, vitamin E and vitamin C in the algal biomass were measured using high performance liquid chromatography (HPLC). Analysis of the three antioxidants was performed on a model HP15 HPLC equipped with a UV detector. Separation and determination were performed on a C18 (ODS) column ( mm). The gradient mobile phase consisted of acetonitrile/chloroform (29:8, v/v), for b-carotene and vitamin E estimation, while in case of vitamin C, the column was eluted

3 World J Microbiol Biotechnol (21) 26: with a mixture of methanol and water (1:1, v/v). Total run time for separations was *15 min at a flow rate of 1 ml min -1. b-carotene, vitamin E, and vitamin C were detected at 254, 47, and 254 nm, respectively (Gertz 199). Statistics Data are expressed as mean ± SE from three independent experiments. One-way analysis of variance (ANOVA) was performed using SPSS version 12, followed by Duncan s test (Christensen 1996). Results Protein and carbohydrate (mg/g dry weight) Dry weight (mg/5ml) Figure 1 shows that over the wide range of tested, the highest value of the dry weight of D. bardawil was obtained at 7.5 (control culture). At extremes of, i.e., 4 and 1, a sharp decline in dry biomass gain was observed (about 47. and 47.5% of control at 4 and 1, respectively). Shifting the value to 6 or 9, the dry weight increased and recorded and 81.9% of control, respectively. Above 1, the growth of D. bardawil was completely arrested. On the other hand, the dry biomass of C. ellipsoidea was significantly enhanced at the alkaline values. The dry weight gain increased to 131.3, 146.7, and 136.9% of control at 9, 1, and 11, respectively. At acidic and slightly acidic values (4 and 6), the dry weight significantly decreased recording 62.3 and 84.% compared to the control (Fig. 2). As shown in Fig. 1, both protein and total carbohydrate of D. bardawil were affected by of the culture medium. Protein and carbohydrate (mg/g dry weight) Dry weight (mg/5 ml) and glycerol (%) Fig. 1 Effect of different values on dry weight (filled circle), protein (filled square), carbohydrate (filled triangle) and glycerol (multiplication symbol) of Dunaliella bardawil after 1 days growth period (mean ± standard error, n = 3) Fig. 2 Effect of different values on dry weight (filled circle), protein (filled square) and carbohydrate (filled triangle) of Chlorella ellipsoidea after 1 days growth period (mean ± standard error, n = 3) Protein and carbohydrate attained their maximum values at 7.5 (control culture). Shifting the value towards the acidic or alkaline side significantly decreased the content of both protein and carbohydrate. The protein content recorded 61.9, 82.8, 74.6, and 64.5%, whereas carbohydrate content reached 69.2, 88.5, 85.1, and 73.7% as compared with control at 4, 6, 9, and 1, respectively. It is worth noting that the lowest values of protein and carbohydrate were obtained at extremes ( 4 and 1). The protein content of C. ellipsoidea attained its maximum value at the acidic value ( 4) reaching 142.5% of control ( 7.5). The increase of to 6 or 7.5 led to a significant and progressive decrease in the protein content. A sharp significant reduction in the protein content of this alga was obtained at the alkaline values. Contrarily, the carbohydrate of C. ellipsoidea attained its highest value at 9 (Fig. 2), at which carbohydrate content recorded 142.5% of the control, then significantly decreased at 1 to a value approximately equal to that of control. The lowest value of total carbohydrate was obtained at 4. On the other hand glycerol production by D. bardawil was significantly reduced at both alkaline and acidic values as compared with that of the control culture. 1 seems to have a more significant effect on glycerol production than the other values. With regard to the pigment accumulation in D. bardawil subjected to a wide range (Fig. 3), 7.5 (control) exhibited the highest accumulation values of chlorophyll a, chlorophyll b and carotenoids. The contents of the three pigments significantly decreased as the shifted towards the acidic or alkaline sides. The reduction in the values of these pigments was less significant at the acidic side. The

4 1228 World J Microbiol Biotechnol (21) 26: Pigment content (mg/g dry weight) Chlorophyll a Chlorophyll b Carotenoids β-carotene (µg/1 mg fresh weight) Vitamin E (µg/1 mg fresh weight) and Vitamin C (µg/g fresh weight) Fig. 3 Effect of different values on pigment content of D. bardawil after 1 days growth period (mean ± standard error, n = 3) lowest values of the three pigments were attained at 1, recording 47.2, 56.9, and 55.4% control for chlorophyll a, chlorophyll b, and carotenoids, respectively. From the data depicted in Fig. 4, the highest values of chlorophyll a, chlorophyll b, and carotenoids occurred at 4, 6, and 7.5. With a shift of to the alkaline side, the contents of the three pigments significantly decreased. 11 recorded the lowest value of chlorophyll a, chlorophyll b, and carotenoids. It is worth noting that the reduction in chlorophyll b content at the alkaline side was more significant than both chlorophyll a and carotenoids. As shown in Fig. 5 both 6 and 9 significantly enhanced the accumulation of b-carotene by D. bardawil, reaching 1.7 times at 6 and more than fourfold the control value at 9. Similarly, the contents of both vitamin E and vitamin C significantly accumulated at 6 and 9, reaching maximum values of and 28.7% Pigment content (mg/g dry weight) Chlorophyll a Chlorophyll b Carotenoids Fig. 4 Effect of different values on pigment content of C. ellipsoidea after 1 days growth period (mean ± standard error, n = 3) Fig. 5 Accumulation of antioxidants, b-carotene (filled circle), vitamin E (filled square), and vitamin C (filled triangle) in Dunaliella bardawil grown in different values for 1 days growth period (mean ± standard error, n = 3) l of control, respectively, in the case of vitamin E and and 265.5% of the control, respectively, in the case of vitamin C. It is worth noting that the enhancement of the three antioxidants (b-carotene, vitamin E, and vitamin C) accumulation by D. bardawil was more highly significant at 9 than at 6. The accumulation of b-carotene by C. ellipsoidea significantly increased at both 6 and 1, recording maximum values of and 172% of control, respectively (Fig. 6). On the other hand, vitamin E significantly increased at 1 reaching of control, whereas 6 had no significant effect on the accumulation of this vitamin in C. ellipsoidea cells. In contrast, the vitamin C content of C. ellipsoidea significantly reduced to 63.7 and 7.1% as compared with control value at 6 and 1, respectively. β-carotene (µg/1 mg fresh weight) Vitamin E (µg/1 mg fresh weight) and vitamin C (µg/g fresh weight) Fig. 6 Accumulation of antioxidants, b-carotene (filled circle), vitamin E (filled square), and vitamin C (filled triangle) in Chlorella ellipsoidea grown in different values for 1 days growth period (mean ± standard error, n = 3)

5 World J Microbiol Biotechnol (21) 26: Discussion The hydrogen ion concentration () of the growth medium influences many processes associated with algal growth, metabolism, and uptake of ions (Borowitzka and Borowitzka 1988). The results of the present study show that the optimum for the growth of D. bardawil is 7.5 (control culture). At this the dry weight attained its maximum value. On the other hand, data concerning C. ellipsoidea revealed that the dry weight gain of this microalga was enhanced at the alkaline side especially at 1. Shifting to the acidic side significantly decreased the dry weight. It may be worth noting that both microalgae can withstand a wide range extending from 4 up to 1 or 11 in case of D. bardawil and C. ellipsoidea, respectively. Generally, it is believed that the optimum differs according to different algal species. McLachlan (1964) mentioned that the optimum for growth of D. tertiolecta was about 6, whereas that for D. salina and D. viridis was about 9 (Loeblich 1972). On the other hand, Massyuk and Yurchenko (1962) reported that D. salina tolerates a wide rang of values between 5.5 and 1. Similarly, Chlorococcum species can grow at between 5.5 and 9 but the optimum was about 8 (Zhang et al. 1997). The inhibition of the growth of both D. bardawil and C. ellipsoidea over 1 can be explained by the fact that at high alkaline, almost no carbon is accessible for the algae because carbonate ion (CO 2-3 ) is the dominant form of inorganic carbon (Falkowski and Raven 1997) and the bicarbonate ion (HCO - 3 ) is the form utilized by the microorganisms (Azov 1982). At present the exact site of CO 2 evolution inside chloroplasts has been a subject of considerable interest. There is increasing speculation that the RUBISCO-containing pyrenoid may also serve as the site of CO 2 evolution (Sültemeyer et al. 1995). It is tempting to speculate that thylakoid inclusions in the pyrenoid may function to create a large proton concentration, which could serve to convert bicarbonate to CO 2 (Badger et al. 1993). There is evidence for localization of carbonic anhydrase in the pyrenoid of C. reinhardtii (Sültemeyer et al. 1995). According to Pronina et al. (1981), a significant amount of bicarbonate (HCO - 3 ) can pass into illuminated thylakoids where the is near 5 and dehydrate to CO 2 in the presence of intrathylakoid membrane-bound carbonic anhydrase. The resulting CO 2 can then escape from the thylakoids into the pyrenoid. A second variant is also possible: the membrane bound carbonic anhydrase of mammalian cells can form membrane channels (Wistrand 1984). Carbon deficiency has previously been shown to increase ROS levels in algal cells and this imposes oxidative stress conditions to the cells. Vardi et al. (1999) found that the production of ROS in the dinoflagellate Peridinium gatunense depended on carbon availability; low carbon concentration stimulated the production of ROS, whereas high carbon concentration decreased ROS in the cells (Choo et al. 24). The data obtained concerning the effect of on both protein and carbohydrate contents of D. bardawil revealed that 7.5 seems to be the most suitable for the accumulation of protein and carbohydrates in Dunaliella cells. At acidic or alkaline values, the contents of both protein and carbohydrates significantly decreased. In the range of studied, 4 recorded the lowest values of both protein and carbohydrates in the cells of this micro alga. Furthermore, the results of the present study showed that the protein content of C. ellipsoidea attained its highest accumulation at 4, then significantly decreased at the other values recording its lowest value at 11. On the contrary, the accumulation of carbohydrate in the same alga enhanced at 9. The lowest value of carbohydrate was obtained at 4. It is worth noting that the accumulation of protein and carbohydrates in D. bardawil is favored at 7.5, while in case of C. ellipsoidea the optimum was 4 and 9 for the accumulation of protein and carbohydrates, respectively. These two studied microalgae could grow between 4 and 1 (for D. bardawil) and 4 and 11 (for C. ellipsoidea). Accordingly, the two algal species could withstand a broad range of, making them suitable candidate for outdoor cultivation without regulation. The above mentioned results are in agreement with other studies. According to Essa (1995), 9 was more suitable for protein production in D. tertiolecta and D. salina, while 6 was suitable for D. bardawil. The same author emphasized that 9 was more suitable than 6 for carbohydrate synthesis in D. salina and D. parva. On the other hand, Taha (22) reported that both D. tertiolecta and D. parva need a lower for protein synthesis. The results obtained further show that glycerol production by D. bardawil was significantly enhanced at 7.5. At the other values studied, the accumulation of glycerol was markedly reduced. This result agreed with the findings of Essa (1995), who concluded that 7.8 is the optimum value for glycerol production in the cells of D. salina and D. parva. The same author also reported that 6 caused a decreased accumulation of glycerol in D. bardawil, D. salina, and D. parva, but the optimum value of glycerol accumulation in D. bardawil was 9. The effect of different values on pigmentation of D. bardawil exhibited a similar trend to that of protein and carbohydrates, i.e., 7.5 also favored the accumulation of chlorophyll a, chlorophyll b, and carotenoids. The content of the three pigments significantly decreased at higher or lower values. On the other hand, the three pigments of C. ellipsoidea markedly accumulated when the alga was

6 World J Microbiol Biotechnol (21) 26: grown at 4, 6 and 7.5 and then decreased in the alkaline range. Abe et al. (1999) reported that reduced resulted in inhibition of chlorophyll synthesis. Wegmann and Metzner (1971) found that the optimum for photosynthesis is generally lower that required for growth. Concerning the accumulation of carotenoids, Del Campo et al. (2) reported that values of 6 9 were found to stimulate carotenogenesis in Muriellopsis species. At extreme acidity ( 4), no carotenoid accumulation was observed in Chlorococcum sp. Increase in increased the total carotenoid yield up to 8 (Liu and Lee 2). This is the case in our results as the pigments of D. bardawil, including carotenoids increased as value increased, reaching the maximum value at 7.5. In contrast, Zhang et al. (1997) concluded that under acid conditions, the cellular content of carotenoids of Chlorococcum sp. was higher than that at other values. The same authors further concluded that the effect of medium on pigmentation could be an indirect effect through growth and physiological changes, rather than a direct effect on the biosynthesis of carotenoids. Several studies have suggested that carotenoids have an important role as antioxidants in microorganisms and oxygen radicals are important inducers for carotenoid biosynthesis (Kakizono et al. 1993). It could be concluded that of the medium might affect the formation of ROS, which in turn would affect the quantity and composition of carotenoids in both D. bardawil and C. ellipsoidea. The data obtained further revealed that the changing of the culture medium s to either acidic ( 6) or alkaline ( 9) values greatly enhanced the accumulation of b-carotene, vitamin E, and vitamin C in the cells of D. bardawil. This enhancement of the three antioxidants was more prominent at 9. In case of C. ellipsoidea, 6 greatly enhanced the accumulation of b-carotene as compared with the alkaline 1. In contrast, vitamin E significantly accumulated at 1, while 6 had no effect on its content as compared with control. The vitamin C content of C. ellipsoidea, on the other hand, was significantly reduced at both 6 and 1. Previously published data on Dunaliella spp. indicated that these algae accumulated massive amounts of carotene under conditions which retard cell division such as extreme values (Abalde et al. 1991). Liu and Lee (2) found that the cellular content of total carotenoids of Chlorococcum sp. cultures were directly proportional to the oxygen level in the culture medium postulating the active oxygen species may involved in the induction of these carotenoids in this alga. It has been suggested that oxidative stress on the algal cells might act as an enzyme activator, an oxidizer for oxygen and hydroxylation of b-carotene or as an H? acceptor for NADP regeneration (Kakizono et al. 1993). In this connection, Liu and Lee (2) reported that the increased carotenoids in Chlorococcum sp. with increasing between 5 and 8, may be explained by hydroxylation activities in that algal species preferring relatively acid conditions, while oxygenation activities in this strain prefer relatively neutral-alkaline conditions. Anon (1983) stated that 9 is the optimal for b-carotene production in D. bardawil, while 6 and 9 were optimal for carotenoid production in D. tertiolecta and D. salina, respectively (Essa 1995). On the other hand, Del Campo et al. (2) observed that maximum accumulation of carotenoids in Muriellopsis sp. was obtained when cells were grown at 6.5, decreasing markedly at higher or lower values. Similarly, in Chlorella zofingiensis large amounts of astaxanthin and canthaxanthin were accumulated at 5 6.5, whereas at 7 8 no carotenoids were found in non-limiting cultures of the same alga (Liu and Lee 2). Conclusion The tested microalgae could grow in a broad range of, making them suitable candidates for open system cultivation without regulation. b-carotene was highly recovered in D. bardawil at 9. b-carotene and vitamin E were greatly accumulated in C. ellipsoidea cells grown at 6 and 1, respectively. References Abalde J, Fabregas J, Herrero C (1991) b-carotene, vitamin C and vitamin E content of the marine microalga Dunaliella tertiolecta cultured with different nitrogen sources. Bioresour Technol 38: Abe K, Nishimura N, Hirano M (1999) Simultaneous production of b-carotene, vitamin E and vitamin C by the aerial microalga Trentepohlia aurea. J Appl Phycol 11: Anon A (1983) Effect of on Dunaliella bardawil biomass and production of carotenoids. 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