Production of ß-carotene and glycerol from Dunaliella bardawil and D. salina isolated from the Egyptian wet -lands Qarun and Bardawil

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1 P Lux International Conference on Ecological, Environmental and Biological Sciences (ICEEBS'212) Jan. 7-8, 212 Dubai Production of ß-carotene and glycerol from Dunaliella bardawil and D. salina isolated from the Egyptian wet -lands Qarun and Bardawil Ola. Taha, Wafaa. Abo El-Kheir, Fayza. Hammouda and Howayda. Abd El-Hady Abstract- The halophytic green alga Dunaliella has an important economic value because it contains ß-carotene, glycerol and other accessory pigments, which have pharmaceutical application. The present study was conducted to optimize some ecological parameters light intensity, and duration, temperature and Nitrogen concentrations for increasing carotene content of the tested Dunaliella species. Two Dunaliella species were used in this study D. bardawil and D. salina. Glycerol and β-carotene contents of the algae were also measured. The two Dunaliella species were cultivated on both natural and artificial media under controlled experimental conditions. For D. bardawil, its ß carotene content increased from 8.15 and 75.55mg/L to mg/l and 1.84 in natural and synthetic media respectively. For D. salina its ß carotene concentration increased from 4.27 mg/l and 2.78mg/L to 6. and 4.47 for both natural and synthetic media respectively. These experiments were carried out under the following experimental conditions: continuous illumination with intensity of о 22.5 X1P 4P PC (±1) glycerol increased from 59. to 8.67and mg/l, for D. salina and from 114. to 11. and 146. mg/l, for D. bardawil grown in synthetic and natural media, respectively. For Nitrogen its concentrations used in artificial medium was reduced by one tenth of its value reported in the standard medium for D. salina and for D. bardawil without any alteration in carotene production. This modification is highly important for large-scale production of ß carotene, by reducing the production cost. D. bardawil contains high contents of ß carotene and glycerol compared with D. Salina. Keywords- Β-carotene, Dunaliella bardawil, Dunaliella salina, Glycerol T I INTRODUCTION HE halo-tolerant green algae Dunaliella bardawil and Dunaliella salina possess the ability to accumulate large amounts of some economically important metabolic compounds as β-carotene (pro-vitamin A) In this study the two species were grown under different stresses to accumulate high concentrations of β-carotene. Ola Ezz El-Din Taha is with National Institute of Oceanography and Fisheries, Egypt. Reference [28] reported that Dunaliella bardawil, a unicellular green alga that can be induced to accumulate massive amounts of β-carotene. [2]. investigated that high irradiance is the most important factor responsible for the massive accumulation of β- carotene by the halo-tolerant green alga, Dunaliella Salina..Referance [6]. studied that the plant carotenoid acts as accessory pigments for photosynthesis, as protection against photooxidation through absorption of light in the blue region of spectrum, and the energy absorbed can be transformed to chlorophyll.reference[8]. mentioned that among algae the only eukaryotic and photosynthetic organism able to grow in media containing a very wide range off salt concentrations from.5% to saturation (around 5%) is Dunaliella. Reference [2] mentioned that Dunaliella tolerate high salinity and high temperature also [2]. stated that the highest accumulation of anti-oxidant vitamins in Dunaliella salina was observed when it is grown under high light intensity in media containing high salt (%) and low nitrogen concentrations (5ppm), they reached] 1.14 % carotenoids 1.2% tocopherol and 2.5% ascorbic acid. Reference [] suggested that in Kuwait there are a number of algal mats (Dunaliella sp.) within various salt marshes (Al Khiran area). Such marches are exposed to extreme environmental conditions, particularly during the summereference [12]. reported that Dunaliella is one of very few microorganisms which can thrive in media containing high salt concentrations thus allows the cultivation of a relatively pure culture. Under appropriate growth conditions (high light intensity and limiting nitrogen supply) a massive amount of β-carotene and glycerol are formed. In addition, six clones of Dunaliella salina were isolated from salt fields in the coast of the Gulf of Thailand. One of the clones produced the highest carotenoid content in modified medium at % NaCl under continuous illumination. Reference [1] investigated why Owens Lake is red? The salt lakes are red due to a dense population of unicellular organism, Dunaliella algae which, produces a red carotenoid pigment, the red pigment completely mask the green of its chloroplast. II. METHODOLOGY Dunaliella bardawil was isolated from the salt marshes of El- Bardawil Lake at the northern coast of Sinai Peninsula at about 5 Km, near El-Arish City. Dunaliella salina was isolated from the salt marshes of Lake Qarun at about 75 Km south west of Cairo city. The two species were purified and identified according to [1] the Systematic position of Genus Dunaliella: is Class: Chlorophyceae Order: Volvocales Family Dunaliellaceae Genus Dunaliella. Isolation and purification were carried out according to the steps reported by 69

2 P lux P lux. P lux, P lux P International Conference on Ecological, Environmental and Biological Sciences (ICEEBS'212) Jan. 7-8, 212 Dubai [24]. Algal cultures were bacteriologically purified by antibiotic treatment recommended by [5]-[18]. and then were checked for purity according to[7]. Dunaliella salina was grown in MH synthetic medium (Loeblich, 1982), which was prepared by dissolving the following salts in 1 liter of glass distilled water:7.5g NaCl; 1.5g MgCl 2.6H 2 O; 1.g KNO ;.4g NaHCO ;.5g KH 2 PO 4 ; 1.89mg EDTA; 2.44mg FeCl.6H 2 O and.61mg H BO. ph was adjusted at 7.5.While Dunaliella bardawil was cultivated in a growth synthetic medium of [1]. which prepared by dissolving the following salts in 1 liter of distilled water: 56g NaCl;.g MgSO 4 ;.4g NaHCO ;.18g CaCl 2 ;.4g KNO ;.16 g KH 2 PO 4 ;.154g FeCl ;.96g EDTA and.5g H BO. ph was adjusted at 7.8. Dunaliella bardawil and Dunaliella salina were grown in natural medium enriched by 4 mg.l KNO ; 26 mg.l KH 2 PO 4 ; 6 mg.l Fe-EDTA. The salinity was adjusted to 15 % by adding the commercial salt, of each lake according to[4]. The culture flasks containing media were autoclaved at 121 C and 1.5 kg cm -2 for 15 minutes. After autoclaving, the culture flasks were left to cool for one day to allow for ph stabilization at 7.5. The sterilized media were inoculated with the stock culture that contain µgl chlorophyll a and mg/l carotene, for Dunaliella salina and with µg/l chlorophyll a and mg/l carotene, for Dunaliella bardawil. Stock and experimental cultures were incubated in a locally made incubator with vigorous hand agitation once a day to prevent clumping of cells. The test duration was 8 days at temperature of 28 ± 1 C with a light program of 12h light followed by 12h darkness (12L: 12D), at light intensity of 7.5 X 1 lux. The measurements of field parameters of Lake Qarun and Lake Bardawil in the flourishing season(summer) of Dunaliella salina and Dunaliella bardawil such as light intensity, temperature, number of cells/ ml, chlorophyll a, b in µg/l ß carotene, in mg /L ph, glycerol and salinity) For pigment analysis 25ml of salt marshes water of both Qarun and Bardawil Lakes were filtered on Whatman Glass fiber filter paper GF/F and ß-carotene was measured according to.[17] by spectrophotometer in all experiments while determination of glycerol was measured only at the optimum condition according to the method reported by 164]. using spectrophotometer The β-content content at different light intensities for the two tested algae. of 7.5, 12.5, 17.5 and 22.5 lux x 1. And at different light durations 12 hours light: 12 hours dark, 14 hours light: 1 hours dark and continuous light and different concentrations of nitrogen. which had been decreased in the (standard natural media St) in D. salina and D.bardawil from.4 g/l potassium nitrate to.1 g/l. (modified natural media Md).while in case of synthetic medium of D. salina potassium nitrate had been decreased from 1g/L to.1g/l and in case synthetic medium for D. bardawil potassium nitrate had been decreased from.4g/l to.1g/l (new modified synthetic media Md) for D. salina and D.bardawil respectively)..glycerol was measured at only the optimum conditions in mg/l. III RESULTS The field data were 5 X 1³ cells /ml for D. baradwil and 1 X 1 ³ cells /ml for D. salina. Carotene content was 1 mg/l and 2.46 mg /L for D. bardawil and D. salina, respectively. Salinity was 4.9 g/l for Lake Qarun and g/l for Lake Baradwil. Glycerol was 114. in D. barawil and 59. mg/l for D. salina The experiment showed that the total carotene of the two tested algae increased with increasing light intensity (Fig 1).Total β carotene increased from 1.27 mg/l at 7.5 X 1P to 2.78 mg/ L at 22.5 X 1P P lux for D. salina in synthetic medium. In natural medium the total β carotene increased from 1.6 mg/l at 7.5 X 1P lux to 4.27 mg/l at light intensity 22.5 X 1P For D. bardawil, in synthetic medium β carotene increased from mg/l at light intensity of 7.51X 1P Plux to 9.82 mg/l at 22.5 X 1P in natural medium, it increased from 8.15 mg/l at 7.5 X 1P to 94.5 mg/l at 22.5 X 1P Plux Total carotenoids (mg l) Fig.1 Effect of different light intensities on total carotenoids of D. salina and D. bardawil in both natural (N) and synthetic (S) at light dark cycle of 12 h L & 12 h D Light intensities (Lux X 1 ) D.bardawil (S) D.bardawil (N) D.salina (S) D.salina (N) The optimum light intensity was accompanied with rise in ο temperature being 4 P PC (±1). The effect of different durations of light atoptimum light intensity of 22.5 X 1³ lux and temperature of 4ºC (±1) for the two tested algae in ß carotene natural and synthetic media was illustrated in (Fig 2). The data revealed that in D. salina grown at 12 h light: 12 h dark the amounted to 2.78 mg/l. At 14 h light and 1 h dark the ß carotene increased to.8 mg/l and reached in continuous light to 4.1 mg/l in synthetic medium while in natural medium β carotene increased from 4.27 to 6.2 mg/l in continuous light. In case of D. bardawil, it increased in synthetic medium from 9.82 to 1.94 mg/l in continuous light, also it increased in natural medium from 94.5 to mg/l. Total carotenoids (mg l ) Fig.2. Effect of different light durations on carotenoid contents of D. salina and D. bardawil in natural (N) and synthetic media (S) 12/12 14/1 contenuous Ligt/dark cycles D.salina (N) D.salina (S) D.bardawil (N) D.bardawil (S) After reaching the optimum conditions for the maximum content of ß carotene (light intensity 22.5X1, temperature 4ºC(±1) and continuous light), experiments were carried out to optimize the concentration of nitrogen. In case of D. bardawil, nitrogen had been decreased in natural medium from.4 to.1 g/l and this slightly increased the amount of carotene from 17 to mg/l. The decreasing of nitrogen concentration in synthetic medium from.4 to.1 g/l, the β-carotene content slightly decreased from 1.94 to 1.84 mg/l. So the decreasing of nitrogen concentration did not significantly affect the amount of ß carotene of D.bardawil. In case of D. salina, the nitrogen used for preparation of the synthetic medium (.1 g/l) was 1 folds lower that used for the standard medium (1 g/l). Under this level of nitrogen the amount of β- carotene slightly increased from 4.1. mg/l in the standard medium to 4.74 in synthetic medium Md). The lowering of nitrogen levels from.4 to.1g/l in natural medium caused a slight increase in β-carotene content of D. salina from 6.2 to 6. mg/l amount of β- The data of Nitrogene for the two tested algae are given in (Fig and Fig 4). 7

3 P day, International Conference on Ecological, Environmental and Biological Sciences (ICEEBS'212) Jan. 7-8, 212 Dubai Total carotene (mg l ) Total carotene (mg l ) natural St media Md media St media Md media Concentrations of major elemnt (Nitrogen, gl ) synthetic Fig.. Effect of different concentrations of Nitrogen on total carotenoids of D. salina in synthetic and natural media natural St media Md media St media Md media Concentrations of major elemnt (Nitrogen, gl ) synthetic Fig. 4. Effect of different concentrations of Nitrogen on total carotenoids of D. salina in synthetic and natural media Glycerol was measured after reaching the optimum conditions, glycerol in case of D. salina increased from 59. to 8.67 mg/l and mg/l in case of synthetic and natural media, respectively. While in case of D. bardawil it increased from 114. mg/l to 11. and 146. mg/l in synthetic and natural media, respectively). D. bardawil, which has the highest in β carotene content and glycerol than D. salina. IV DISCUSSION In a comparison study between Lake Bardawil and Lake Qarun, the results revealed that the physical and chemical analysis of salt marshes of Lake Bardawil as well as Lake Qarun revealed a high salinity values in both salt marshes of the two lakes. No remarkable difference was observed in light intensity of the salt marshes of both lakes. Salt marshes of both lakes had no appreciable difference in the temperature. The observed data of the high salinity, high light intensity and high temperature seem to be favorable for both Dunaliella salina and Dunaliella bardawil in the two salt marshes and they have the ability to accumulate ß-carotene.[2]. They reported that Dunaliella is halotolerant and capable of growing in.5 M NaCl and above. The optimum temperature was higher at higher light intensity. In this connection [12], found that Dunaliella spp accumulate large amount of β-carotene when cultivated under appropriate conditions including high light intensity, high sodium chloride concentrations, nitrate deficiency and extreme temperature [12]. found that Dunaliella is one of the very few microorganisms which can survive in media containing high salt concentrations (6.12 % NaCl) for optimal growth. It is note worthy that[14]. reported that salt is the major factor of the Dunaliella growth medium and cells reaches its optimum growth in 5 % NaCl. [2]. found that the high irradiance and nutrient stress are extremely important factors in the development of larger cells of D. salina containing larger quantities of β-carotene. Reference []. reported increase in β-carotene concentrations per cell of D. salina with increase in salinity. The present study modified the artificial media to contain few and less quantities of dissolving salts giving the same amount of ß- carotene. Natural waters of both lakes waters were enriched by inorganic Nitrogen + phosphorus. This enrichment resulted in a significant increase in different growth parameters. These results supported with the findings of [19].. The results also revealed that both D. salina and D. bardawil were th harvested on the 8P P. Day which contained the maximum carotene contents.reference [2]. give one week as the residence time of D. salina to produce the maximum content of β-carotene. He explained this period to be the required time for development of the largest sized cells, which contain highest concentrations of β- carotene. Our results agree also with the finding of [6]. who th reported that at 8P the cells of the D. salina are at their exponential phase of growth and so harvested at this time. Total carotenes considerably increased with increasing in light intensities and duration This view agrees with the findings of [4[,The increase in the cellular level of β-carotene with increasing in both light intensities and duration of light could be explained according to[28]. who attributed this increase to: a) an acceleration rate of biosynthesis of β-carotene due to enhanced activity or synthesis of carotenogenic enzymes; b) continued β-carotene synthesis in non dividing cells, or a reduced rate of its degradation or secretion; c) activation of genes involved in β-carotene accumulated. An increase in total carotenes with increasing light duration agrees with [2]. who observed a long residence time's influence an increase of β-carotene production by D. salina by developing layers cells containing higher concentrations of B- carotene.reference [25]. attributed an increase in β-carotene with increasing light intensity to increasing in lipid globules of D. bardawil which induced by high light intensity to produce 2- fold increase in the content of β-carotene globules. The analysis of Dunaliella spp from both studied Lakes by spectrohphotometer revealed that D. bardawil contains much more concentrations of carotenes (1 mg LP P) than D. salina (2.46 mg LP P). Under optimum culture conditions, D. bardawil proved also to be more successful species in β-carotene production yielding (1.84.) and mg LP P) than D. salina and 6. mg L) in both synthetic and natural media. There was a decrease in chlorophyll a,b by the increase in light intensities and time of exposure this agree with the findings of [11]. they reported a negative relation between chlorophyll content and β-carotene in D. bardawil. They found that the β-carotene accumulation coupled with chlorophyll depletion under high light intensities. The obvious degradation of chlorophyll contents of algae by high light intensities can be attributed to photo destruction of chlorophyll by excess irradiance, thus carotenoides act as a protective against photo inhibition by screening chlorophyl[12]. and during carotenogensis the concentration of chlorophyll decreases in D. salina cells. Chlorophyll concentrations, which have been decreased by increase light intensity, may be an intracellular signal for the induction of B-carotene synthesis[21]. Moreover, [29]. reported that when D. bardawil grown under sunlight spectrum with high light intensities, chl a/b ratios was decreased combined by 6% increase in β-carotene, they further concluded that light is a limiting factor influence on pigment synthesis. It is generally believed that in D. salina and D. bardawil chlorophyll a and b content decreased with increasing in both light intensities and duration of light. On the other hand, the carotenoid synthesis showed a different trend, since the content increased with increasing light intensities and longer time of exposure. 71

4 International Conference on Ecological, Environmental and Biological Sciences (ICEEBS'212) Jan. 7-8, 212 Dubai Reference[19]. confirmed this finding and[2]. he stated that chlorophyll a and b of D. salina and D. bardawil decreased at high light intensities. D. salina produces a red carotenoid pigment under the blistering summer heat which completely masks the green chlorophyll pigment of its chloroplast[1]. At optimum conditions of continuous light, light intensity of 22.5 x 1 lux, 4 C (±1) and nitrogen deficiency, the maximum values of glycerol was found in the cells of D. bardawil when grown on natural media The ratio of glycerol and carbohydrate synthetic media, respectively, and 4:1 and 9:1 in natural media, respectively. This indicates that glycerol synthesis suppressed carbohydrate by nearly 4:11 times.reference[8] also confirmed the accumulation of glycerol on the expense of carbohydrates. He found that in Dunaliella, the carbohydrate synthesized during photosynthesis was consumed for glycerol synthesis especially in saline media. Reference [22]. reported that light induced conversion of starch into glycerol by the unicellular, halotolerant green algae D. tertiolecta and D. salina.also these data agree with [9]-[1]. The maximum glycerol production by alga D. salina was recorded in fluorescent light, this reported by[7] who observed an increase in glycerol in red, blue and green light and less in the dark. It is generally believed that under stress conditions such as high light intensity or nutrient starvation, cell of the unicellular alga D. bardawil overproduce and accumulate B-carotene in newly formed triacylglycrol droplets. When the synthesis of triacylglycerol is blocked, the overproduction of β-carotene is also inhibited, thus the formation of these structures and β-carotene are interdependent[]. The data of this investigation also revealed that temperature of 4 C (±1) was suitable for glycerol and ß-carotene accumulation in D. salina and D. bardawil cultures in synthetic and natural media. The results in this investigation regarding temperature and light intensity were depending on each other, temperature increase gradually by increasing light intensity and gave the same changing in the biochemical parameters. Temperature is an important factor, which affect the growth of algal cells. Dunaliella spp have a wide temperature tolerance. The halophytic D. salina has been reported to survive at temperature ranging from 5 C to about 4 C [9]. Moreover, unicellular alga Dunaliella survive in the blistering summer heat and concentrated brine is truly remarkable [1]. Reference [4], reported increase in illumination and temperature causes a significant increase in the rate of β-carotene synthesis in D. salina reaching maximal value at 22 C. The optimum temperature of several Dunaliella spp was higher at high light intensity than at lower intensity. The optimum temperature to synthesize carotene in Dunaliella was C [1]. observed the same marked increase in β-carotene synthesis and maximal specific growth rates of D. bardawil and D. salina around C. At temperature above 4 C the growth of Dunaliella was sharply decrease and was completely inhibited at temperature exceeding 45 C. Similar observation of B-carotene of D. salina and D. bardawil under extreme temperature and increase in the β-carotene to chlorophyll ratio were reported by [11].Reference [2]. reported that D. salina isolated from Thailand produce the highest carotenoids content under continuous illumination of 27 µ mol m - 2 s and temperature of -7 C Reference [12]. suggested that the optimal growth temperature for Dunaliella is around 2 C, glycerol retention by the cells is strongly temperature-dependent, Above 25 C, the rate of glycerol release into the medium gradually increase and above 4 C it increases dramatically, this due to the effect of temperature on membrane organization. Reference[19]. found that 28 C was the optimum temperature for the growth of Dunaliella spp (D. salina, D. bardawil, D. parva and D. terricola) and D. bardawil was the most resistant algal towards high temperature and contains the highest values of carotenoids, carbohydrate and protein. In this connection, [26]. found that at optimum temperature 28 C induced an optimal growth conditions, high content of chlorophyll, β-carotene, a specific thylakoid fatty acids and triacylglycerol (TAG) of D. salina cells. Data of the preliminary tests gave maximum yield of carotenoids for both D. salina and D. bardawil in media contain KNO than NaNO. This fact coincides with that of [15]. The results obtained from optimization of carotenoids synthesis indicated that the carotenoids of both tested algae increase with gradual decrease in nitrogen concentrations reaching the maximum values for D. salina at.1 and.1 g/l in synthetic and natural media, respectively and.1 g/l for D. bardawil at both synthetic and natural media, this was confirmed by [2]-[27]. The response of carotenoid synthesis by D. salina and D. bardawil to decrease in nitrogen concentrations differed from the other biochemical parameters (chlorophyll, lipid, protein and carbohydrate) which decreased with decreasing nitrogen concentrations. Accumulation of carotenoids under conditions of nitrogen starvation has been previously documented in green micro algae Dunaliella spp.[4]. who found that excluding nitrogen from the nutrient medium leads to tripling of the B-carotene in D. salina cells as compared with the control. In addition, decreasing concentrations of nitrate, decreasing the chlorophyll concentrations more markedly than the other parameters and thus produce redder D. bardawil cells (carotenoid to chlorophyll ratios increased from 2 to 9). Phosphates limitation did not affect the relative concentrations of the chlorophyll and carotenoids component[16]. This observation contrasts the finding of [4]-.[5]b. who reported that carotenoids biosynthesis and accumulation were not stimulated but inhibited when D. salina cells were grown in KSP medium under low nitrate concentrations. The accumulation of carotenoids in algal cells grown under nitrogen starvation may be due to stimulation of lipolysis to produce acetyl COA that serves as a precursor for synthesis of carotenoids.reference[19]. suggested that in enriched Lake Bardawil water with low concentrations of N and P, the glycerol content of D. bardawil was highly increased, whereas D. salina synthesizes fewer amounts. The primary goal of the present study was the biomass production of Dunaliella spp. In order to accomplish this objective, many experiments were designed and carried out. The general approach during the experimentation was to subject the selected algae to the conditions, which favor the production of the highest biomass coupled with the highest yield of the desired by-products (β-carotene). The experiments acquired the optimum conditions for β- carotene production as: continuous light intensity (22.5 x 1 Lux), temperature of 4 C (±1), ph and nitrogen starved media. These conditions achieved a mean concentration of mg L in synthetic and natural media respectively. D. bardawil, which was the highest in carotene content than D. salina.the new modified natural and synthetic media for the two tested gave the same amount of β-carotene. REFERENCES [1] Armustrong.W.P.: Why Owens Lakes are red.hydrob. Vol.12 pp [2] Abd El-Baky, H.H.. Biochemical studies on some algae as a source of antioxidant vitamins. Ph.D. Thesis, Dept. of Biochemistry, Fac. of Agriculture. Cairo University [] Al-Hasan, R.H. and Sallal, A.J. Preliminary studies on a halo-tolerant algae Dunaliella sp. from Kuwait salt marches. J. Univ. Kuwait (Sci.).Vol. 12 pp [4] Araneda, P.; Jimenez, C. and Silva, B.G... Micro algae from northern Chile. III Growth and β-carotene content of three isolates of Dunaliella salina from the Atocma desert. Rev. Biol. Mar. Valparaiso.Vol. 27 no (2). pp

5 International Conference on Ecological, Environmental and Biological Sciences (ICEEBS'212) Jan. 7-8, 212 Dubai [5] Araneda, P.; Jimenez, C. and Silva, B.G.Micr algae from northern Chile.growth from in low coast media of two isolates of Dunaliella salina.esud.oceonal Vol.11 pp [6]Bartly,G.E. and Scolnik,P.A.Plant caratenoids pigments for photoprotection, visual attraction and human health. The Plant cell. Vol.7 pp [7] Bekheet, I.A. and Syrett, P.J.. Urea-degrading enzymes in algae. British phycological Journal Vol.12, pp [8] Ben-Amotz, A. The Beta-carotene accumulating halo-tolerant alga Dunaliella. Biotechnology. Vol.7 no.(1) pp [9] Ben-Amotz, A. and Avron, M. Glycerol, β-carotene and dry algal meal production by commercial cultivation of Dunaliella. In algae Biomass, ed. G. Shief & C.J. Soeder, pp Amsterdam, ElSevier, North Holland Biomedical press. [1] Ben-Amotz, A. and Avron, M.. The potential use of Dunaliella for production of glycerol, β-carotene and high-protein feed. In Biosaline Research. Alook to the future, ed. A., San Pietro, pp , New York; Plenum Publishing Corporation. [11] Ben-Amotz, A. and Avron, M.. On the factors which determine massive β -Carotene accumulation in the halo-tolerant alga Dunaliella badawil. Plant. Physiol., Vol.72 pp [12] Ben-Amotz, A. and Avron, M.. The wave length dependence of massive carotene synthesis in Dunaliella bardawil (Chlorophyceae). J. Phycol., Vol.25 pp [1] Ben-Amotz, A. Adaptation of the unicellular alga Dunaliella parva to a saline environment. Journal of phycology. Vol no.11 pp [14] Ben-Amotz, A.; New mode of Dunaliella biotechnology two phase growth for β-carotene production J, Applied phycol.vol.7pp [15] Borowitzka A.M. and Borowitzka J.L. Dunaliella In " Micro-algal biochology", ed., New York pp [16] Chitlaru, E. and Pick, V.. Selection and characterization of Dunaliella salina mutants defective in halo-adaptation. Plant Physiol., Vol.91 pp [17] Davies, B.H.. Analysis of cartenoid pigments. In chemistry and biochemistry of plant pigments by Goodvin, T.W. pp [18] Droop, M.R.. A procedure for routine purification of algal cultures with antibiotics. Britieh phycological Bulletin, Vol. pp [19] Essa, A.M.. Osmoregulatory metabolites accumulated in halophilic algae grown on organic wastes. M.Sc. Thesis, Depart. Of Botany, Fac. of Sci. Cairo University [2] Ginzburg, M.. Dunaliella a green alga adapted to salt. J. Botanical research. Vol.14 pp [21] Gomez-Pinchetti, J.L.; Ramazanov, Z.; Fontes, A. and Garcia-Reina, G.. Photosynthetic characteristics of Dunaliella salina in relation to betacarotene. J. Appl. Phycol., Vol.4 no. (1) pp [22] Goyal, A.; Brown, A.D. and Gimmler, H.. Regulation of salt-induced starch degradation in Dunaliella tertiolecta. J. Plant Physiol., Vol.127 pp [2] Grobbelaar, J.U.. Influence of a real density on β-carotene production by Dunaliella salina. J. Appl. Phycol., Vol.7 pp [24] Hoshow, R.W. and Rosowski, J.R.. Methods for microscopic algae. In., J.R. Stein (ed.) Hand book of phycological Methods, culture method and growth measurements. Cambridge Univ. Press, Cambridge, pp [25] Jimenez, C. and Pick, U.. Differential stereoisomer composition of beta-carotene in thylakoids and in pigment globules in Dunaliella. J. Plant physiology., Vol.14 no. () pp [26] Klyachkogurvich, G.L.; Pronina, N.A.; Furnadzhiera, S.; Ramazonov, Z.M. and Petkov, G,. Lipid composition and membrane state of Dunaliella salina cell subjected to sub-optimal temperature. Russian Journal of Plant Physiology. Vol.44 no. (2) pp [27] Laurel, and Loeblich, A. A planospores of Dunaliella salina (Chlorophyta). J. Protozology./ Vol.16 pp [28] Lers, A.; Biener, Y. and Zamir, A.. Photoinduction of massive β - carotene accumulation by the alga Dunaliella bardawil. Plant Physiol., Vol. 9 pp [29] Levy, H.; Tal, T.; Shais, A. and Zamir, A.. The photosynthetic response of Dunaliella bardawil to the changes in the sunlight spectrum under field conditions. J. Biol. Chem., Vol.268 pp [] Loeblich, L.A.. Photosynthesis and pigments influenced by light intensity and salinity in the halophilic Dunaliella salina (chlorophyta). J. Mar. Biol. ASS.U.K., Vol. 62 pp [1] Massyuk, N.P. Morfologica, Sistemetika, Ekologija, Geograficeskoe Rasprostranenieroda Dunaliella teod., Naukova Dumka, Kiev pp. 244, [2] Powtongsook, S.; kittakoop, P.; Menasveta, P. and Wisessany, S.: Isolation and characterization of Dunaliella Salina from Thailand.J. Applied Phycology, Vol 7 no. (1-2) pp [] Rabbani, S.; Beyer, P.; Volinting, J.; Hagueney, P. and Kleining, H.. Induced beta-carotene synthesis driven by triacylglycerol deposition in the unicellular alga Dunaliella bardawil. Plant Physiol., Vol.116 no. (4) pp [4] Semenenko, V.E. and Abdullaev, A.A.. Parametric control of β - carotene biosynthesis in Dunaliella salina cells under condition of intensive cultivation. Plant Physiol., Vol.27 no. (1) pp [5] Speucer, C.P.. On the use of antibiotics for isolating bacteria-free culture of marine phytoplankton organisms. Journal of Marine Biology. ASS.U.K., Vol. 1 pp [6] Taha, H.M.. Massive production of some essentially important metabolic compounds in Dunaliella Salina. Masc. Thesis Dept. of Botany, Fact. Of Sic., Alexandria University [7] Taker, A. and Kumar, H.D.. Effects of ph, Light quality and various carbon sources on glycerol production by the brackish water alga Dunaliella salina. Cytobios., Vol.9 no. (61) pp [8] Wegmann, K. Osmotic regulation of photosynthetic glycerol production in Dunaliella. Biochemica et Biophysica Acta, Vol. 24 pp [9] Wegmann, K.; Ben-Amotz, A. and Arron, M.. Effect of temperature on glycerol retention in the halo-tolerant algae Dunaliella and Asteromonas. Plant Physiol.,Vol. 66 pp [4] Yamaoka,Y.: Takimura, O.: Fuse.H. and Kamimura, K.β-Carotene production y Dunaliella salina in led-batch and semi-continuous cultures under nutrient supplement seibutsu-kogaku Kaishi., Vol. 72 no. (2).pp (1994). 7