GROWTH AND ACCUMULATION OF TOTAL CAROTENOIDS IN TWO
STRAINS OF Dunaliella salina Teod. (CHLOROPHYCEAE) FROM
THE NORTHERN AND CENTRAL COAST OF PERÚ

CARLA P. AGUILAR^*, MARIELA GONZÁLEZ, ANA S. CIFUENTES and MARIO SILVA

Departamento de Botánica, Facultad de Ciencias Naturales y Oceanográficas,
Universidad de Concepción, Casilla 160-C, Concepción, Chile.
E-mail: caguilar@imarpe.gob.pe
(Received: March 28, 2002 - Accepted: October 14, 2003)

ABSTRACT

In order to know the dynamics of growth and accumulation of total carotenoids in two strains of Dunaliella salina from the northern (Colán) and central (Chilca) coast of Perú, both strains were cultured during 40 days in controlled laboratory conditions, at 25±2°C, with 150 mol m^-2 s^-1, without aereation. The strains were grown in two culture media with two different salinities: a modified Johnson medium with addition of 15 and 23% NaCl and a Provasoli enriched seawater medium with 12.5 and 20% NaCl. A period of exponential growth during the first 5 days was observed in both strains, followed by a slow linear growth phase and a stationary phase which lasted until the end of the experiment (day 40). The maxima cellular densities were obtained in the modified Johnson medium at 15% NaCl in both strains, being higher in Chilca (2.2x10⁶ cell mL^-1) than in Colán (1.6x10⁶ cell mL^-1). The accumulation of total carotenoids per volume was similar in both strains but different in both media; the highest values were obtained in the Provasoli medium with 20% NaCl (13.3± 2.9 mg L^-1, Colán and 10.6± 2.5 mg L^-1, Chilca). On the contrary, the total content of carotenoids per cell was different in both strains and media; the highest values were obtained in the Provasoli medium with 20% NaCl (110.9± 0.7 and 58.7± 1.3 pg cell^-1 in the strains Colán and Chilca, respectively).

Key words: Total carotenoids, Chilca, Colán, Dunaliella salina, Perú.

INTRODUCTION

The genus Dunaliella is a chlorophycean alga known for being unicellular, biflagellate and devoid of cell wall, which comprises marine, haline and hyperhaline species. In this last group D. salina, a highly resistant organism is found which has been adapted to grow in extreme conditions, such as high light radiations, high salt concentrations, dryness and extreme temperatures. These conditions are typical in desertic, semiarid and/or arid regions of the world, biotopes where this species normally inhabits almost without competitors. Due to the wide salinity gradients observed in these environments, this alga is considered an halophytic and/or an halotolerant species (8, 9, 18).

Another important physiologically characteristic of D. salina is its capacity to accumulate large quantitites of b -carotene (more than 10% of the algal weight) which has this, turned it into highly commercial algal source (7, 10,13, 20, 27,). Nowadays, it is being massively cultured in different parts of the world such as Australia, Israel, China, EUA (10) and also recently, in Chile (29).

In Perú, the researchs on the genus Dunaliella, specifically on D. salina, have been performed on strains isolated from the samples, that were collected in the coastal and southern highlands of the andean region. These researchs dealt with ecological, physiological and life cycle aspects (1, 2, 5, , 24, 25, 26), and they have shown the phenotypic plasticity of the species, as well as them great ability to adapt themself to different saline environments along the national territory.

The Peruvian coastal region is known for being a narrow semidesertic strip with soils with abundant quantities of mineral salts, mainly sodium, calcium and magnesium. The northern coast receives a high solar radiation all year long, and the water temperatures fluctuate between 22°C and 37°C. The central coast, on the contrary, presents a more variable climate, being tropical in summer and with precipitations (drizzle) in winter. The temperatures fluctuates between 19°C and 23°C in winter, and can reach 34°C in the Chilca Salines, in summer. The greatest number of ponds, lagoons and hyperhaline aquatic environments near the littoral, are found along this zone (2, 26).

Considering the high physiological variability reported at a specific level in D. salina (3, 4, 11, 12, 13, 22), the objective of the present research was to study the dynamics of growth and accumulation of total carotenoids in two strains coming from the salines of Colán and Chilca (northern and central coast of Perú, respectively), cultured in controlled laboratory conditions, and to infer its potential use for commercial scale b -carotene extraction.

MATERIAL AND METHODS

Biological material

Dunaliella salina Colán strain was collected in July 1997 from the Colán Salines (81° 04´ LO and 5° 59´ LS, Departament of Piura), at a water temperature varying from 33 to 37°C and 320 to 350%o of salinity. D. salina Chilca strain was collected in January 1999 from the Chilca Salines (76° 42´ LO and 12° 32´ LS, Department of Lima), in water at 34°C and 350 - of salinity.

Growth experiments and total carotenoids quantification.-

Prior to the growth experiences, monoalgal cultures of both strains were stablished. Then, they were grown in two culture media: a Modified Johnson: (J/1) (10), an artificial medium, with addition of 15% and 23% NaCl (J/1+15% and J/1+23%, respectively) and a Provasoli Enriched Seawater: (PES) (21), a natural enriched medium, with 12.5% and 20% NaCl (PES+12.5% and PES+20%, respectively). The concentrations of NaCl in both media were approximately equivalent. The composition of the media is detailed in the Table 1. Each strain was cultured in 200 mL volume from an initial cellular density of 10⁴ cells mL^-1. Cultures were grown in a culture chamber at 25± 2°C, under a constant photonic flux density of 150 m mol m^-2 s^-1, for 40 days.

Table. 1: Nutritional composition of the 2 media employed in the cultures of D. salina, each one supplemental with two different concentrations of NaCl: 12.5% and 20 % of NaCl in Provasoli enriched seawater medium (PES) and 15% and 23% of NaCl in modified Johnson medium (J/1).

In order to study the interaction between the factors (strains and culture media) on all the variables (total carotenoids per volume (mg L^-1), total carotenoids per cell (pg cell^-1) and total carotenoids to chlorophyll "a" ratio (g g^-1)), a factorial experimental design with 2 factors was employed: considering two strains and four media, with 3 replicates for each treatment.

The growth curves (cell density vs. time) were determined by cell counts, using a 1 mL Utermöhl chamber and a Zeiss inverted microscope. Growth rate was estimated in the exponential phase according to the formula:

k= 3.322 x log(N2/ N1)	[div day-1]
(t2- t1)

Where: t₁-t₂= time interval, N₁=cellular density at t₁, N₂= cellular density at t₂, The reciprocal of k is the duplication time in days (Td=1/k) (16).

Total carotenoids and chlorophyll "a" content were determined spectrophotometrically from 90% acetone cell extracts according to the methodology of Wegmann and Metzner (32).

The results were statistically analyzed with the computational program Statistical N°6, using Analysis of Variance and the Tukey test.

RESULTS

Growth curves obtained for each treatment were very similar in both strains (Figs. 1a- b). All the cultures presented an exponential phase of growth during the first 5 days, followed by a stationary phase of growth in PES medium and by a linear growth, which lasted until the end of the experiment (day 40), in J/1 medium.

a)
	Fig. 1: Growth curves of the two strains of D. salina: a) Colán and b) Chilca, cultured in Provasoli natural enriched medium: PES+12.5%; PES+20% and in modified Johnson artificial medium: J/1+15%; J/1+23%, grown at a photon flux density of 150 m mol m-2 s-1 and a temperature of 25± 2°C, during 40 days.
b)

In both strains, the highest growth rates occurred in PES+12.5% medium. The growth rates of the Colán strain fluctuated from 0.37div day^-1 (J/1+23%) to 0.93 div day^-1 (PES+12.5%), and the Chilca strain, from 0.34 div day^-1 (J/1+23% NaCl) to 0.96 div day^-1 (PES+12.5%). Both growth rates were similar in both strains and media. However, the maxima cellular densities observed in PES+12.5% NaCl medium (5.1x10⁵ cells mL^-1 and 9.4x10⁵ cells mL^-1, Colán and Chilca strains, respectively) were much lower than those obtained in J/1+15% NaCl medium (1.6x10⁶ y 2.2x10⁶ cells mL^-1, Colán and Chilca strains, respectively) (Table 2).

Table 2: Growth rate (k) estimated in the logarithmic phase (time interval indicated), maximum average cellular density during the stationary phase grown (cells Max.) (SD = standard deviation), of 2 strains of D. salina from Perú, cultured in Provasoli medium (PES) and modified Johnson medium (J/1) at two salt concentrations as indicated in percentage (NaCl %).

The average content of total carotenoids per volume was similar in both strains, when cultured in the same medium, but different in both media (Fig. 2). However, the total carotenoids content per cell varied significantly (P<0.05) among strains and media (Fig. 3). The Colán strain presented the highest content of total carotenoids both per volume and per cell (Figs. 2 -3). In both strains, the highest total carotenoids content per volume and per cell was obtained in PES + 20% NaCl (13.3 y 10.6 mg L^-1 and 110.9 and 58.7 pg cell^-1, in the Colan and Chilca strains, respectively).

Fig. 2: Maximum average content of total carotenoids per volume (mg L-1), found in 2 strains of D. salina on day 40 of culture. Culture conditions as indicated in legend of fig:1, n=3, ± DE.

Fig. 3: Maximum average content of total carotenoids per cell (pg cell-1), in 2 strains of D. salina, on day 40 of culture. Culture conditions as indicated in legend of fig:1, (symbols as in Fig. 2. n=3, ± DE).

The average content of total carotenoids per volume increased significantly until day 30, in both strains in PES medium (Fig. 4). Only in the Colán strain, cultured in PES+20% NaCl, this parameter continued increasing slightly, after day 30, until the end of the experiment, varying from 11.26 mg L^-1 to 13.3 mg L^-1. In J/1+15% NaCl, on the contrary, both strains showed a sustained increasement of the total carotenoids content, duplicating the amount accumulated in the same medium at 23% NaCl (Fig. 5). In all conditions, the highest concentration of total carotenoids per volume occurred at the end of the experiment (day 40), in both strains.

Fig. 4: Accumulation of total carotenoids per volume (mg L-1) of 2 strains of D. salina, cultured in Provasoli natural enriched medium (PES); Colán strain: PES+12.5% CO, PES+20% CO; Chilca strain PES+12.5% CH, PES+20% CH, culture conditions as indicated in legend of fig: 1, (n=3, ± DE).

Fig. 5 : Accumulation of total carotenoids per volume Cartv (mg L-1) of 2 strains of D. salina, cultured in modified Johnson artificial medium (J/1); Colán strain J/1+15% CO, J/1+23% CO; Chilca strain J/1+15% CH, J/1+23% CH, culture conditions as indicated in legend of fig: 1, (n=3, ± DE).

The total carotenoid content per cell, on the contrary, did not show the same pattern as the total carotenoids per volume. The maximum content (110.9 pg cell^-1) ocurred on day 40 in the Colán strain, when grown in PES+20% NaCl. In this condition, the Chilca strain cells reached a maximum carotenoids content of 58.69 pg cell^-1 on day 30 and after this value began to decay. Both strains also presented the maximum content of total carotenoids on day 30, when grown in PES+12.5% NaCl (Fig. 6). On the other hand, the maximum quantities of total carotenoids per cell in J/1 medium were always obtained earlier than in PES medium: 50.63 pg cell^-1 on day 20 and 40.66 pg cell^-1 on day 10, for the Colán and Chilca strain, respectively, both at 23% NaCl. At 15% NaCl, both strains showed quantites of carotenoids per cell lower than 15 pg cell^-1 on day 10 (Fig.7).

Fig. 6: Accumulation of total carotenoids per cell (pg cell-1) of 2 strains of D. salina, in Provasoli natural enriched medium (PES); Colán strain PES+12.5% CO, PES+20% CO; Chilca strain PES+12.5% CH, PES+20% CH. Culture conditions as indicated in legend of fig: 1. (Symbols as in Fig. 4, n=3, ± DE).

Fig. 7: Accummulation of total carotenoids per cell (pg cell-1) of 2 strains of D. salina, in modified Johnson artificial medium (J/1); Colán strain J/1+15% CO, J/1+23% CO; Chilca strain J/1+15% CH, J/1+23% CH, Culture conditions as indicated in legend of fig: 1. (Symbols as in Fig. 5, n=3, ± DE).

The total carotenoids to chlorophyll "a" ratio showed significantly different (P<0.05) behavior patterns in both media (Figs. 8 and 9). In PES, this ratio increased until the end of the study in both strains (Fig. 8). In J/1, on the contrary, this ratio increased abruptly only during the first 10 days reaching a value which was maintained until the end of the experiment (Fig. 9). The highest ratios found in PES+20% NaCl, in both strains were 28.8 g g^-1 and 23.1 g g^-1 for the Colán and Chilca strains, respectively.

Fig. 8: Variation of the total carotenoids to chlorophyll "a" ratio (g g-1), in 2 strains of D. salina, cultured in Provasoli enriched seawater medium (PES); Colán strain PES+12.5% CO, PES+20% CO; Chilca strain PES+12.5% CH, PES+20% CH, Culture conditions as indicated in legend of fig: 1. (Symbols as in Fig. 4, n=3, ± DE).

Fig. 9: Variation of total carotenoids to chlorophyll "a" (g g-1), in 2 strains of D. salina, in modified Johnson artificial medium (J/1); Colán strain J/1+15% CO, J/1+23% CO; Chilca strain J/1+15% CH, J/1+23% CH Culture conditions as indicated in legend of fig: 1. (Symbols as in Fig. 5, n=3, ± DE).

DISCUSSION

The dynamic of growth in D. salina is a function of the environmental and/or experimental conditions to which the alga is exposed (14,15). Therefore, the comparison of the results obtained in this study with those reported for other strains of D. salina (4, 11,13, 30, 22) has restrictions relative to the differences in experimental conditions and in the history of the inoculum. In relation to this last point, the present research was carried out soon after the algae were isolated from field samples; therefore, it can be inferred that they were not acclimated enough to the laboratory conditions.

In this research, both strains exhibited the maximum growth rates at the lowest salinity, in both media, similar to the results found by Cifuentes et al. (12) and Miravalles and Leonardi (22), for a Chilean (CONC-006) and an Argentinian strain, respectively, both under similar culture conditions.

The significantly higher growth rates of both strains in PES medium, compared with those obtained in J/1 medium (0.9 and 0.6 div day^-1 and 0.96 and 0.5 div day^-1 for the strains Colán and Chilca, respectively), are coincident with the results found by Cifuentes et al. (11,13). The higher carrying capacities of the cultures at the end of the experiment obtained in J/1 medium were in PES medium (1.6x10⁶ and 5.1x10⁵ cells mL^-1 and 2.2x10⁶ and 9.4x10⁵ cells mL^-1 for Colán and Chilca, respectively) are in agreement with the results reported by the above mentioned authors.

The highest total carotenoids contents per unit of volume were estimated in PES+20% NaCl and J/1+15% NaCl (Figs. 2, 4 and 5). Although in this last condition, the carotenoids per cell content was minor than in PES+20% NaCl, the higher carrying capacities of the cultures in J/1+15% NaCl than in PES+20% NaCl, explain the similitude between the carotenoids contents in both conditions.

Both strains were similar in their average total carotenoids content per volume in both conditions, but they differed significantly in their maximal total carotenoid content per cell. This can be related to the differences found in the mean cell size among both strains (20.01± 1.2mm x 14.75± 2.5mm Colán and 15.29± 1.1mm x 13.12± 1.9mm Chilca) and probably to the intrinsic physiological characteristics of the Colán strain which comes from ponds exposed at higher levels of irradiance than the Chilca strain.

The comparison among the average values of total carotenoids per volume obtained in the strains of this research with those reported by Cifuentes et. al., (12) and Gómez et. al., (17) for Chilean strains, conclude that Chilean strains CONC-001, CONC-006 and CONC-007 are more carotenogenics, per volume unit, than the Peruvian strains. However, in terms of total carotenoids per cell, the Colán strain is superior to the talassohaline coastal strain CONC-001 from Antofagasta and D. bardawil from Israel and similar to the highland andean strains CONC-006 and CONC-007 from the Salar of Atacama (12,17). The Colan strain is also superior to the Thailandese coastal strains (30) cultured in J/1+20% NaCl (10.7 to 32.8 pg cell^-1), but at slightly higher temperatures and irradiances.

The Colán strain showed the highest total carotenoids to chlorophyll "a" ratio. This ratio, as was expected, increased with the salinity of the medium, similar to the results of Cifuentes et al. (11,12,13).

When comparing the carotenogenic parameters estimated for both strains, it became evident that the Colán strain was more carotenogenic than the Chilca strain. This can be explained through the environmental differences between both sites of collection. The Colan strain comes from salines near the equatorial line, with tropical climate and exposed to very high average solar radiations and temperatures. The Chilca strain, on the other hand, comes from the central coast where the climate is variable and the solar radiation and temperatures are lower than in Colán (2).

In general, it is known that strains exposed to high irradiation synthetize and accumulate high quantity of carotenoids as a protection mechanism in front of the oxidative stress, detrimental to the cell (6, 31, 19, 28). Even though the level of total carotenoids found in both Peruvian strains is enough to consider them as potential b -carotene producers, more research is needed in order to improve the optimal growth conditions and to determine the proportion of b -carotene in the total content of carotenoids.

ACKNOWLEDGMENTS

The authors wish to thank biologists Giovanna Vera and Víctor Hugo Vera for their help in collecting samples and Mrs. María Dávila for her help in the experimental part of this research and Dr. Jorge Tam for greatly improved the English in the manuscript. This work was supported by cultures of microalga laboratory and phytochemical laboratory of Concepción University.

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