QUANTITY AND QUALITY OF b-CAROTENE PRODUCED BY TWO
STRAINS OF DUNALIELLA SALINA (TEODORESCO 1905)
FROM THE NORTH OF CHILE

PATRICIA I. GOMEZ, MARIELA A. GONZALEZ *AND JOSE BECERRA

Departamento de Botánica, Facultad de Ciencias Naturales y Oceanográficas, Universidad de Concepción, Casilla 160-C, Concepción, Chile.
(Received: June 6, 1999 - Accepted: August 26, 1999)

SUMMARY

The growth and the quantity and quality of carotenes produced by two Chilean strains of D. salina (CONC-001 and CONC-007), grown in two different media (PES and SM) were analyzed. No important differences were found between the two media either in the growth or in the carotenogenesis suggesting that the low-cost media (SM) would be appropriate for large scale culture of D. salina. The strain CONC-007 displayed the lower growth rate, but the highest productivity of carotenoid per cell and the highest 9-cis b-carotene content whereas the strain CONC-001 exhibited the highest growth rate but a lower accumulation of carotenoid per cell and a low 9-cis b-carotene content. The Chilean strains were both more carotenogenic than the reference strain Dunaliella bardawil.

KEY WORDS: D. salina strains, physiological variability, b-carotene isomers, culture media.

RESUMEN

Se analizó el crecimiento así como la cantidad y calidad de los carotenos producidos por dos cepas chilenas de D. salina (CONC-001 y CONC-007) cultivadas en dos medios de distinta formulación (PES y MS). La composición del medio de cultivo no afectó el crecimiento ni la carotenogénesis de las cepas analizadas, sugiriendo que el medio de bajo costo (MS) podría ser apropiado para el cultivo de D. salina a mayor escala. La cepa CONC-007 presentó la menor tasa de crecimiento pero la mayor productividad de carotenoides por célula así como el mayor contenido de 9-cis b-caroteno, el cual corresponde al isómero de mayor interés comercial. Por otro lado, la cepa CONC-001 mostró la mayor tasa de crecimiento pero una menor acumulación de carotenoides por célula y un bajo contenido de 9-cis b-caroteno. Ambas cepas chilenas resultaron ser más carotenogénicas que la cepa de referencia Dunaliella bardawil.

PALABRAS CLAVES: Cepas de D. salina, variabilidad fisiológica, isómeros de b-caroteno, medio de cultivo.

*To whom correspondence should be addressed.

INTRODUCTION

The halotolerant microalga Dunaliella salina Teodoresco has the capacity to accumulate high quantities of b-carotene (more than 10% of the algal dry weight) (7,5), for which it is cultured massively for commercial purposes in Australia, Israel, United Sttes, China (12,13) and recently in Chile (Gattavara S.A. Company, Iquique).

The carotenes of D. salina are a mixture of b-carotene and of the b.carotene isomers, 9-cis and all-trans (7,8). By contast, synthetic b-carotene is composed only of the all-trans isomer (10,6).

The high liposolubility of the 9-cis b-carotene permits its great absorption and storage in animal tissues (3). In addition, studies in vitro and in vivo on the b-carotene antioxidant activity of D. salina have demonstrated that the 9-cis isomer would have greater antioxidant effect than the all-trans isomer, and a therapeutic effect in the prevention of diseases that involve the production of free radicals is suggested (17,9). Hence, the natural b-carotene fulfills the requirements of some specialized markets, which results in its much higher price than the synthetic pigment (10). Based on the above mentioned background and considering the interest that the massive cultivation of D. salina as a natural source of b-carotene engenders, new strains that present a high carotenogenic capacity, that is, a high b-carotene content, a high productivity of the 9-cis b-carotene isomer and a low a-carotene content would be desirable. This latter, due to the lack of information on the effect of a high consumption of such carotene on human health.

Parra et al. in 1990 (20,21), reported the presence of D. salina in Chile; since then, eight strains of this species have been isolated, and are maintained in the Microalgae Culture Collection at the University of Concepción, Chile. These strains come from the North of the country: CONC-001 from "Laguna La Rinconada" in Antofagasta (20,21) and the other seven (CONC-003 until CONC-009) from "Salar de Atacama" (14). The many physiological studies carried out on these strains revealed a great variability in growth rate and in the accumulation of total carotenes when cultivated under different conditions, such as medium composition (1,2,15,16), salinity range (14,15) and irradiation intensity (2). On the other hand, Markovits et al. (18) analyzed the composition in carotenes produced by the strains CONC-001 and CONC-006, finding a significant variability of the b-carotene yield, as well as in the isomers of b-carotene. Even though the content and yield of b-carotene can be manipulated by the culture conditions in D. salina (8,4), it has been demonstrated for the Chilean srains that this is possible only within certain limits, since undoubtedly, the nature of the strains themselves has a fundamental effect on the quantity (14,15,16) as well as on the quality (18) of the carotenes produced.

The objective of the present study is to increase present-day knowledge of the physiological variability found among native strains of D. salina with industrial potential. For this purpose, the carotenogenic capacity of the strains CONC-001, CONC-007 and D. bardawil were analyzed (this latter, as a reference strain), by cultivating them in two media of different formulation (PES and SM), but with the same salinity (high) and nitrogen concentration (low). These media differ mainly in that PES medium was prepared with high reagent grade, while the macronutrients of the SM medium were obtained from agricultural fertilizers and salts of domestic use, being therefore, a very cheap medium. Since high salinity and low concentration of nitrogen have been demonstrated to induce carotenogenesis in the Chilean strains of D. salina (1,2,14,15,16), the quality of the rest of the components of the medium should not affect the carotenogenic capacity of the strains.

The results revealed that the composition of the medium in general, does not affect the carotenogenic capacity of the strains. The most carotenogenic strain was CONC-007, which presented the greater accumulation of carotenoids per cell together with the highest proportion of 9-cis b-carotene, making it potentially usable from a commercial point of view.

Biological material

In this study unialgal cultures of two Chilean strains of D. salina, CONC-001 and CONC-007, were used. Strains CONC-001 is from "Laguna la Rinconada", Antofagasta (23°39'S; 70°24'W) and strain CONC-007 from "Salar de Atacama" (23°30'S; 68°15'W). Both strain are maintained in the Microalgae Culture Collection, University of Concepción. Dunaliella bardawil (ATCC 30861) from Lake Bardawil, Israel, was also included.

Culture

The strains were cultured in two natural enriched media: i) Provasoli sea water enriched medium (19) with a supplement of 12.5% NaCl and ii) simple medium for Dunaliella (3) modified and supplemented with 12.5% NaCl (Table I). The Provasoli medium (PES) was prepared with high reagent grade, while in the simple medium (SM) the macronutrients were supplied from agricultural fertilizers and salts of domestic use. The latter was used to study the algal response to a low cost and easy formulation medium, being of interest for industrial level culture. Both media had the same salinity and contained 9.1 mg l^-1 of nitrogen (limiting concentration).

Due to operational limitations, only two replicas of each strain were cultured in 275 mL of each medium (PES and SM). The inocula of the 3 strains were obtained from cultures growing in modified artificial Johnson medium (11) with 15% NaCl, which provides abundant nutrients. The concentration of 15% NaCl in the Johnson medium is equivalent to a supplement of 12.5% NaCl in PES and SM media. The initial cell density was 1 x 10⁴ cells mL^-1. Cultures were maintained with aeration, at a continuous photon flux density of 180 µmol m^-2 s^-1 from fluorescent day light tubes and at 30 ± 2°C. The growth rate, k (divisions day^-1), was evaluated during the logarithmic phase of growth as in Cifuentes et al. (15).

Complete to volume with filtered sea water

Pigments quantification

The total carotenes and chlorophyll content were determined in 28 day-old cultures (stationary phase) by spectrophotometry from extracts in 90% acetone (22). The extracts from total carotenes were analyzed by HPLC using a chromatograph Vari-Chrom/VA-Varian with UV-Visible detector. A stainless steel column of 15 cm x 3.9 cm i.d. packed with C18 reversed phase material of 5 µm particle size was used. Elution was performed with isocratic solvent of 1 mL min^-1 methanol tetrahydrofurane (95:5 v/v) and its detection utilized a wavelength of 450 nm. The injection volume was 20 µL.

All the results correspond to the average values obtained from two replicas analyzed.

RESULTS

Dunaliella salina strain CONC-001 showed the highest growth rates (divisions day^-1) in PES as well as in SM media (k: 0.71 and 0.66, respectively) while the strain CONC-007 showed the lowest growth (Table II).

After 28 days of culture, the strain CONC-007 and D. bardawil yielded more caretonoids per culture volume in the SM medium than in PES medium. The values obtained in SM were 12.0 and 7.4 mg l^-1 for CONC-007 and D. bardawil, respectively, versus the 10.2 and 5.5 mg l^-1 obtained in the PES medium. On the other hand, the strain CONC-001 yielded more carotenoids per culture volume in PES medium than in SM (Table II).

With respect to carotenoids per cell, the strain CONC-007 was the most carotenogenic, with 53.7 and 50.0 pg cell^-1 in PES and SM respectively, while the less carotenogenic was D. bardawil with 14.5 and 13.5 pg cell^-1. Likewise, the strain CONC-007 exhibited the highest carotenoids/chlorophyll "a" ratios and D. bardawil the lowest ones (Table II). Although CONC-001 accumulated much lower carotenoids per cell than CONC-007, its higher growth rate in both media resulted in a productivity per culture volume (mg l^-1) that was very similar for both strains (Table II).

The quality of carotenes also varied in the three strains analyzed. The strains CONC-001 and CONC-007 showed a similar percentage (20%) of a-carotene in both media; on the other hand, a-carotene was not detected in Dunaliella bardawil (Figure 1 and Table III). The two Chilean strains of D. salina presented notable differences in the proportion of the b-carotene isomers: the strain CONC-001 accumulated a greater proportion of the all-trans isomer while the strain CONC-007 showed a greater proportion of the 9-cis isomer. The highest 9-cis/alll-trans ratio (3.8) was produced by the strain CONC-007 cultured in PES. Dunaliella bardawil presented a very similar content of both b-carotene isomers (Figure 1 and Table III).

FIG. 1. Pigment analysis by HPLC of synthetic all-trans b-carotene and of extracts of Dunaliella salina, strains CONC-001 and CONC-007 and D. bardawil cultured in PES. Retention times: a-carotene = 16.7 min (1); all-trans b-carotene = 17.9 min (2); 9-cis b-carotene = 18.6 min (3).

-------- ND: non detected

DISCUSSION

One of the economic factors that must be evaluated when considering algal biomass production for commercial purposes, is the cost of the culture medium to be used (1). Even though some low-cost media have been demonstrated to be appropriated to induce carotenogenesis in some native strains of D. salina (1,2), until now no experimental data existed on the effect of the quality of the nutrients on the composition of the carotenes accumulated by these strains. As demonstrated in this research, the composition of the culture media employed did not affect notably the production of total carotenoids (Table II) nor the quality of the accumulated pigments (Table III), in the analyzed strains. The finding is biotechnologically very interesting, since it demonstrates that a medium of low cost and easy formulation, such as SM, can be used to culture D. salina at massive scale, without affecting negatively the quality of the pigment produced by the strain.

The results of growth rates and total carotenoids accumulation in the strains CONC-001 and CONC-007 agree well with those of Cifuentes et al. (14,15,16). They found that among all the Chilean strains of D. salina isolated until now, CONC-001 exhibits the highest growth rate and the lowest total carotenoids content per cell, while CONC-007 presents a low growth rate, but has shown to be the most carotenogenic strain under all the culture conditions employed. However, until now there was no information about the quality of the pigment accumulated by the strain CONC-007. In the present study it is demonstrated that, in addition to accumulating large quantities of total carotenoids per cell (Table II), the quality of the b-carotene produced by this strain is optimum since it contains a high proportion of the 9-cis isomer (Table III) and this isomer is of great interest for some specialized markets (10). In spite of the low growth rate that this strain shows when cultured at a laboratory scale, its yield could be higher if it is cultured at a greater scale. Future research should focus on pilot scale cultures in order to evaluate the real feasibility of using this strain for commercial purposes.

Dunaliella bardawil accumulated practically equal quantities of the 9-cis and all-trans b-carotene isomers which agree well with what has been found by Ben-Amotz et al., (7) for this taxon. On the other hand, Markovits et al. (18) found that D. salina strain CONC-001 accumulated a higher proportion of the all-trans than the 9-cis b-carotene isomer. Even though the culture conditions used in the present investigation were not the same as those employed by Markovits et al. (18), we also detected a greater accumulation of the all-trans b-carotene isomer in this strain (Table III). These results corroborate that although the culture conditions can affect the production and quality of the pigment produced by D. salina, the nature of the strain also has a fundamental role in its physiological behavior.

The strain CONC-001, in contrast with CONC-007, exhibits a high growth rate, resulting in a carotenoids yield per culture volume similar to that of CONC-007 (Table II). However, as the b-carotene accumulated by CONC-001 contains a higher proportion of the all-trans isomer, this strain is less attractive to be cultured at an industrial scale.

Alpha-carotene was not detected in D. bardawil (Table III). Apparently, this pigment is present in very low amount in this strain, since even though it appears in the chromatograms published by Ben-Amotz et al. 1982 (7), the authors do not mention it. On the contrary, the a-carotene present in both Chilean strains accounts for 20% of the total carotenes (Table III) and would be unfavorable if someone wants to use their pigments in the nutritional industry, since no studies exist on the effect that a high consumption of a-carotene may have in animal organisms (18).

Finally it is worthwhile to point out that in spite of the geographical proximity of the two Chilean strains of D. salina included in this study, they presented notable differences in their physiological behavior.

ACKNOWLEDGMENTS

The authors wish to thank Mrs. Ana S. Cifuentes for her valuable advising in the carotenogenesis experiments, Mrs. María Dávila and Mr. Juan Pablo Carreño for their help in the experimental part of this research, and to Dr. Annette Coleman for greatly improved the English in the manuscript. This work was supported by FONDECYT N 1970990 granted to Dr. Mariela A. González.

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