In the present study Dunaliella sp. that could grow in the Johnson medium was isolated from hypersaline Lake Tuz and its b-carotene production was studied in ...
Ó Springer 2005
World Journal of Microbiology & Biotechnology (2006) 22: 183–189 DOI 10.1007/s11274-005-9017-0
Effect of pH, light intensity, salt and nitrogen concentrations on growth and b-carotene accumulation by a new isolate of Dunaliella sp Abuzer C¸elekli1 and Go¨nu¨l Do¨nmez2,* 1 Department of Biology, Faculty of Arts and Sciences, Abant Izzet Baysal University, 14280, Bolu, Turkey 2 Department of Biology, Faculty of Science, Ankara University, 06100, Bes¸ evler, Ankara, Turkey *Author for correspondence: Tel.: +90-312-2126720-1137, Fax: +90-312-2232395, E-mail: gdonmez@science. ankara.edu.tr Received 8 May 2005; accepted 30 June 2005
Keywords: b-carotene, Dunaliella sp., hypersaline lake, microalgae, production
Summary In the present study Dunaliella sp. that could grow in the Johnson medium was isolated from hypersaline Lake Tuz and its b-carotene production was studied in a batch system, in order to determine the optimal conditions required for the highest b-carotene accumulation. In the experiments with light intensity, the cell numbers and b-carotene content were maximum at pH 9, with 20% of NaCl concentration and 48 kerg cm)2 s)1 light intensity. At this light intensity, the b-carotene content of Dunaliella sp. ranged between 0.177 and 1.095 mg/ml for the culture broth and 0.119 to 0.261 ng/cell on a per cell basis under the nitrogen limitation. At the end of the experiments, the maximum b-carotene accumulation and the cell number were obtained at pH 7, 5 mM NaNO3 and 20% NaCl concentrations as 0.261 ng/cell, 4.2106 cell/ml, respectively.
Introduction Unicellular green algae of the genus Dunaliella (Chlorophyta) are ovoid in shape, lack a rigid cell wall, contain one large cup-shaped chloroplast, and are motile with two equal long flagella (Borowitzka & Borowitzka 1992). Dunaliella are probably the most halotolerant eukaryotic organisms known, showing a remarkable degree of adaptation to a variety of salt concentrations from as low as 0.2%, to salt saturation (about 35%); it is the only eukaryotic photosynthetic organism that has been detected in significant numbers in concentrated saline lakes (Ginzburg & Ginzburg 1985; Ben-Amotz & Avron 1990; Ben-Amotz 1991). Dunaliella contains species which possess the unique ability to accumulate large amounts of b-carotene (more than 10% of the dry weight) under proper inductive conditions. The conditions optimal for carotene biosynthesis include high light intensity, high salt concentration, extreme temperatures or nitrate deficiency. Most of the accumulated b-carotene, mainly consisting of the 9-cis and all-trans isomer, was concentrated in intrachloroplastic lipoidal globules, which could be isolated and purified (Ben-Amotz et al. 1982; Ben-Amotz & Avron 1983; Ben-Amotz et al. 1988; Borowitzka et al. 1990).
b-Carotene is currently used as a food coloring agent and as pro-vitamin A in animal food, as an additive to cosmetics, multivitamin preparations and health food products (antioxidant and anti-cancer agent), and in the medical treatment of diseases (Ben-Amotz & Avron 1990). To date, more than 90% of commercial b-carotene is chemically synthesized from petrochemicals. The synthetic b-carotene has the all-trans form while the natural molecule is a mixture of cis (mainly 9-cis and 15cis) and trans isomers. Dunaliella strains may reach 60% of cis, under selected growth conditions. Dunaliella b-carotene stereoisomeric mixture is better adsorbed by living oganisms (Borowitzka & Borowitzka 1992) than the all-trans form. The ability to grow at very high salt concentrations, and the high cell content of b-carotene, glycerol (>50%) or protein (>40%) made this microalga an attractive candidate for commercial production. Although Dunaliella is a unique source of b-carotene, its commercial application is still limited because of low productivity of the culture (Hejazi & Wijffels 2004). Therefore, it will be important to find Dunaliella strains in production areas, where environmental conditions are favourable for outdoor cultivation. The purpose of the present work was to study the potential biotechnological use of a strain of
184 Dunaliella sp. isolated from a hypersaline lake in Turkey.
A. C¸elekli and G. Do¨nmez carried out in triplicate. All determinations were made daily for the incubation period. Analytical methods
Materials and methods Isolation and identification Samples (100 ml) uptake from Lake Tuz (Turkey) in May 1999 was centrifuged and spread (0.1 ml) on Petri plates containing modified Johnson’s medium (Johnson et al. 1968) and incubated at 20±2 °C under continuous illumination (cool-white fluorescent, 32 kerg cm)2 s)1). The composition of the growth medium is NaCl, 100– 250 g; MgCl2 6H2O, 1.5 g; KCl, 0.2 g; CaCl2 2H2O, 0.2 g; NaNO3, 1.5 g; NaHCO3, 0.043 g; KH2PO4, 0.035 g; Fe solution (Na2EDTA, 189 mg/l; FeCl3 6H2O, 244 mg/l), 10 ml and trace element solution (H3BO3, 61.0 mg/l; (NH4)Mo7O24 4H2O, 38.0 mg/l; CuSO4 5H2O, 6.0 mg/l; CoCl2 6H2O, 5.1 mg/l; ZnCl2, 4.1 mg/l; MnCl2 4H2O, 4.1 mg/l), 10 ml in 1l. Agar (12 g/l) was added for plates. The pH of the growth medium was adjusted to 7.5 by dilute (0.01 M) and concentrated (1 M) sulfuric acid or sodium hydroxide solutions. Cells from microcolonies on these plates were isolated by micromanipulation and purified to aseptic conditions by streaking the cells repeatedly on the modified Johnson’s medium agar plate with 25% (w/v) of NaCl. The purified algal cells were transferred to liquid media which was tested for bacterial contamination by plating on bacteriological media.
Determination of b-carotene production In the experiment, first of all the optimum pH for cell growth and b-carotene production was obtained at 10, 15 and 20% (w/v) NaCl concentrations. The pH value of the modified Johnson’s media was adjusted to 6, 7, 8 and 9. For the experiments, 1 ml activated algal culture was inoculated in 100 ml modified Johnson’s media at the same pH and NaCl concentration. In the other experiments, the effect of light intensity on growth and b-carotene accumulation of Dunaliella sp. was investigated. At the optimum pH values for 10, 15 and 20% NaCl concentrations, Dunaliella sp. grew on 48 kerg cm)2 s)1. At the optimum pH values and light intensity, the effect of nitrogen concentration was investigated at four NaNO3 concentrations (1, 5, 10, 17 mM) at 10, 15 and 20% NaCl concentrations. For these experiments, 1 ml of activated culture was grown in 100 ml liquid media at the same pH, NaCl and nitrogen concentrations. A series of batch culture experiments in unshaken flasks illuminated by cool white fluorescent lamps was carried out. The algal cultures were incubated in 250-ml Erlenmeyer flasks at 20±2 °C under continuous illumination for 39 days. In the tests, control cultures without algae were prepared. Each experiment was
For the extraction of b-carotene, 2 ml of algal culture was taken daily from each flask. The cells were pelleted by centrifugation (Hettich EBA-12) at 4000 rev/min for 12 min at room temperature and then resuspended in 5 ml of 80% acetone. Cellular debris was removed by centrifugation at 4000 rev/min for 12 min into a screwcap tube. The concentration of b-carotene in the supernatant was determined spectrophotometrically at 455 nm wavelength using standard curves (Ben-Amotz & Avron 1983). Absorbance measurements were made by using a Shimadzu UV 2001 model spectrometer. Algal growth was monitored by counting cells numbers in a counting chamber (Thoma hemocytometer, 0.1 mm deep). Systatistic analysis The effect of four selected variables (pH, salt, nitrogen concentrations, and light intensity) on the b-carotene production of Dunaliella sp. was analyzed by using Analysis of Variance (ANOVA) fallowing Zar (1999).
Results In the study, the isolated and purified ovoid algal strain, which has one cup-shaped chloroplast and two equal flagella without cell wall, was identified as a Dunaliella species by morphological examination under a microscope (Borowitzka & Borowitzka 1992). The b-carotene accumulation properties of this strain were investigated in a batch system at different pH, light intensity, NaCl and nitrogen concentrations. The results are given as the number of cells per ml (cell/ml) and accumulated b-carotene concentration on a per cell basis (ng/cell) or per ml of culture broth (mg/ml). Effect of initial pH on the b-carotene production at various NaCl concentrations The effect of medium pH value on the b-carotene production at the end of the incubation period was indicated in Table 1. The experiments were performed at various initial pH (6, 7, 8 and 9) values and NaCl (10, 15 and 20% w/v) concentrations. As shown in Table 1, b-carotene content and the cell number of Dunaliella sp. ranged from 0.053 to 0.382 mg/ml and 1.2–4.2106 cell/ ml after 39 days at all tested NaCl concentations. At 15% NaCl concentration there was no significant difference (P>0.05) in the b-carotene content and the cell number between the pH values tested. On the other hand, when b-carotene content and the cell number were differed significantly depending on the effect of salinity concentrations at 10–20% NaCl (P0.05) in cell number with effect of pH values at all NaCl concentrations. In the experiment performed with pH 6, 7 and 8, Dunaliella sp. showed high b-carotene accumulation and cell numbers at 15% NaCl concentration. At these pH values, b-carotene content and the cell number decreased at 20% NaCl concentration. However, the effect of pH 9 was the opposite at pH 6, 7 and 8. At pH 9, bcarotene accumulation increased with a increase in NaCl concentration and the maximum accumulation was obtained at 20% NaCl concentration as 0.382 mg/ml. The increase in salinity from 10 to 20% had little effect on cell numbers at pH 9. At 10 and 20% NaCl concentrations, the cell numbers reached was relatively higher at pH 9 than the other pH values. The maximum cell
number was obtained at 15% NaCl concentration during all the experiments. The accumulated b-carotene concentration on a per cell basis by Dunaliella sp. at different pH and NaCl concentrations during the incubation period is shown in Figures 1, 2 and 3. As seen from Figure 1, b-carotene accumulation at 10% NaCl concentration started after a lag period of approximately 16 days at all pH values. At pH 6, 7 and 8, similar results were obtained at first 27 days period. Between 20 and 37 days, the accumulation of b-carotene increased rapidly, reaching a maximum after about 39 days at pH 7 as 0.061 ng/cell. The lowest b-carotene production was determined at pH 6 as 0.044 ng/cell. At 10% NaCl concentration, the maximum b-carotene
Figure 1. The accumulated b-carotene concentration on a per cell basis by Dunaliella sp. at different pH values and 10% NaCl concentration during the incubation period (T: 20±2 °C; 32 kerg cm)2 s)1 illumination).
Figure 2. The accumulated b-carotene concentration on a per cell basis by Dunaliella sp. at different pH values and 15% NaCl concentration during the incubation period (T: 20±2 °C; 32 kerg cm)2 s)1 illumination).
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Figure 3. The accumulated b-carotene concentration on a per cell basis by Dunaliella sp. at different pH values and 20% NaCl concentration during the incubation period (T: 20±2 °C; 32 kerg cm)2 s)1 illumination).
production was obtained at pH 9 as 0.068 ng/cell during the whole incubation period. In samples containing 15% NaCl concentration b-carotene accumulation was found to be similar at all pH values tested (Figure 2). At all pH values, b-carotene accumulated with a high rate between 15 and 35 days and then continued with a much slower accumulation rate throughout the rest of the experiment. The highest b-carotene production was obtained at pH 9, followed by 8, 6 and 7 with 0.081, 0.078, 0.074 and 0.067 ng/cell respectively. Concordant results were also obtained in the samples containing 10 and 20% NaCl concentrations (Figures 1 and 3) where the highest b-carotene accumulation was obtained at pH 9 during the incubation period. At 20% NaCl concentration, Dunaliella sp. showed similar accumulation rates at pH 6, 7 and 8. In the experiments at pH 9, the b-carotene content obtained was significantly higher (P
