Journal of Biotechnology 76 (2000) 51 59 www.elsevier.

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Carotenoid content of chlorophycean microalgae: factors determining lutein accumulation in Muriellopsis sp. (Chlorophyta)
Jose A. Del Campo, Jose Moreno, Herminia Rodrguez, M. Angeles Vargas *, Joaqun Rivas, Miguel G. Guerrero
Instituto de Bioqumica Vegetal y Fotosntesis, Consejo Superior de In6estigaciones Cientcas-Uni6ersidad de Se6illa, Centro de In6estigaciones Cientcas Isla de la Cartuja, Americo Vespucio, s/n, E-41092 Se6ille, Spain Received 17 March 1999; received in revised form 13 July 1999; accepted 23 July 1999

Abstract Fifteen strains of chlorophycean microalgae have been investigated with regard to their carotenoid prole. Lutein, b-carotene and violaxanthin were present in virtually all of the strains, lutein, in general, being the most abundant carotenoid, whereas canthaxanthin and astaxanthin were found in some strains only. Chlorella fusca SAG 211-8b, Chlorococcum citriforme SAG 62.80, Muriellopsis sp., Neospongiococcum gelatinosum SAG B 64.80 and Chlorella zongiensis CCAP 211/14 exhibited high lutein levels, the latter strain containing in addition substantial amounts of astaxanthin. Muriellopsis sp. was further characterized, since besides a high lutein content (up to 35 mg l 1 culture), it had the highest growth rate (up to 0.170.23 h 1) and maximal standing cell density (up to 8 1010 cells l 1 culture). These levels of lutein are in the range of those reported for astaxanthin in Haematococcus and for b-carotene in Dunaliella, microalgae of recognized interest for the production of these carotenoids. Lutein content of Muriellopsis sp. increased during the exponential phase of growth, with the highest value being recorded in the early stationary phase. Maximum levels of lutein in Muriellopsis sp. cultures were recorded at 20 40 mM NaNO3, 2 100 mM NaCl, 460 mmol photon m 2 s 1, pH 6.5 and 28C, conditions which were, in general, also optimal for cell growth. Growth-limiting conditions, such as pH values of 6 or 9 and a temperature of 33C, were found to stimulate carotenogenesis in Muriellopsis sp. This strain represents a potential source of lutein, a commercially interesting carotenoid of application in aquaculture and poultry farming, as well as in the prevention of cancer and diseases related to retinal degeneration. 2000 Elsevier Science B.V. All rights reserved.
Keywords: Batch culture; Carotenoids; Chlorophycean microalgae; Lutein

1. Introduction The achievement of efcient systems for the generation of carotenoids represents a major issue in the eld of microalgal biotechnology. The po-

* Corresponding author. Fax: +34-5-94460065. E-mail address: avargas@cica.es (M.A. Vargas)

0168-1656/00/$ - see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 5 6 ( 9 9 ) 0 0 1 7 8 - 9

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tential of microalgae as a commercial source of these pigments is widely recognized (Borowitzka, 1988, 1992; Callegari, 1989). In the microalgae, carotenoids function as accessory pigments in the photosystems, as structural components of light harvesting complexes, as well as photoprotective agents, also playing roles in phototaxis (Hagen et al., 1993; Taylor, 1996; Eskling et al., 1997). Only a small number of carotenoids have found commercial application, and these include b-carotene, lycopene, astaxanthin, canthaxanthin and lutein. They are used as food dyes, as feed additives in aquaculture and to enhance the pigmentation of chicken and egg yolks. Sales of lutein as a feed additive in the United States amount to about $150 million per year (Johnson and Schroeder, 1995). Regulations on the use of synthetic dyes in the food industry are currently very stringent. This has stimulated research and development of the production and use of carotenoids from microalgae as food additives. Carotenoids also have applications in cosmetic industries (Borowitzka, 1988, 1992; Benemann, 1992; Johnson and Schroeder, 1995). Carotenoids have also been proposed as effective preventive agents for a variety of human diseases. b-Carotene, lutein and zeaxanthin are claimed to display cancer preventing properties (Richmond, 1990; Le Marchand et al., 1993; Ziegler et al., 1996; Vitamin E Research and Information Service, 1997). In addition, intake of lutein has been strongly correlated with a decreased risk of cataracts and age-related retinal degeneration (Jacques et al., 1988; Rock et al., 1994; Seddon et al., 1994). Only two microalgae are presently recognized as commercial sources of carotenoids, namely the halophilic green agellate Dunaliella salina, which accumulates b-carotene (Avron and Ben-Amotz, 1992), and the freshwater green alga Haematococcus plu6ialis, which accumulates astaxanthin (Boussiba and Vonshak, 1991; Chaumont and Thepenier, 1995). Available information about production of interesting carotenoids by other microalgae is scarce, although several strains of Chlorophyceae have been reported to accumulate canthaxanthin, astaxanthin and/or b-carotene (Borowitzka, 1988; Arad et al., 1993).

This work deals with the screening of 15 strains of chlorophycean microalgae on the basis of their content in some carotenoids of commercial interest and cell growth. The effect of several nutritional and environmental factors on the levels of these pigments has been studied in the particular strain Muriellopsis sp., in order to derive optimum conditions for lutein accumulation.

2. Materials and methods The strains of chlorophycean microalgae used in this work were: Chlorella fusca 211-8b and 211-8c, Chlorococcum citriforme 62.80, Coelastrum proboscideum var. dilatatum 217-2 and var. gracile 217-3, Muriella aurantiaca 249-1, Muriella decolor 249-2, Neospongiococcum gelatinosum B 64.80, Tetracystis aplanosporum 91.80, Tetracystis intermedium 94.80 and Tetracystis tetrasporum 98.80, from the Culture Collection of Gottingen Univer sity (Germany); Chlorella zongiensis 211/14 and 211/51, from the Culture Collection of Algae and Protozoa of the Institute of Freshwater Ecology in Ambleside (UK); Muriellopsis sp. and Tetracystis sp., were isolated from the Emporda marsh in Cataluna (Spain) and from the Amerigo river in Antarctica, respectively, by Professor M.C. Hernandez Marine (Botany Laboratory, Univer sity of Barcelona, Spain), who also classied these strains, which were puried to axenic culture by our group. Muriellopsis sp. was classied according to Broady (1982), and Tetracystis sp. to Bourrelly (1990). These strains are available at the authors laboratory, and cultures will be provided upon request. Cells were grown photoautotrophically by bubbling through the cell suspension air supplemented with 1% (v/v) CO2 as the source of carbon at a ow rate of 100 l l 1 culture h 1. The culture medium of Arnon et al. (1974), modied to contain 4 mM K2HPO4, was used. Unless otherwise indicated, the medium was supplemented with 20 mM NaNO3 as the nitrogen source. For the experiments aimed to determine the effect of NaCl on lutein content, the medium, containing 2 mM NaCl, was supplemented with NaCl 35200 mM, as indicated. The microalgae

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were grown in batch culture at 28C, in 4.5 cm wide 0.2-l capacity asks, laterally illuminated with mercury halide lamps at 460 mmol photon m 2 s 1, except where indicated, measured at the surfaces of the culture containers. To evaluate the effect of pH on growth and carotenoid content, Muriellopsis sp. was grown in a Braun Biotech photochemostat equipped with a 2-l air-lift reactor, and illuminated at 560 mmol photon m 2 s 1 (uorescent compact lamps, Philips PL-T 32 W/840/4p). Air at a rate of 33 l l 1 culture per h was bubbled through the cell suspension. The corresponding pH value was maintained by injection of CO2 pulses through the cell suspension when the pH value rose over the xed set point. For experiments at pH 9, the culture medium was supplemented with 50 mM NaHCO3. For dry weight determinations 5-ml aliquots of the cell culture were ltered through Whatman GF/C paper, washed three times, and the lters containing the algae were dried at 80C for 24 h. Cell number was determined using a Coulter counter, model Z1. Specic growth rate (m) was calculated from the measured cell number, during the logarithmic phase of growth, using the equation: v= ln x2 ln x1 t2 t1

using a programmable photodiode-array detector (Waters 991). Standards of astaxanthin, canthaxanthin and b-carotene were kindly provided by Hoffmann-La Roche, Switzerland. Violaxanthin was a gift from Dr L. Lubian, Instituto de Cien cias Marinas, CSIC, Cadiz, Spain. Lutein and b-carotene were supplied by Sigma (St Louis, MO).

3. Results and discussion The carotenoid content of cultures of 15 chlorophycean microalgae, either from culture collections or isolated from natural environments is shown in Table 1. Data on biomass in the cultures is also provided. In all strains, with the exception of C. zongiensis CCAP 211/14, lutein was the most abundant carotenoid. The highest lutein levels (2338 mg l 1 or 0.21 pg cell 1) were found in C. fusca SAG 211-8b, C. citriforme, N. gelatinosum and Muriellopsis sp. Violaxanthin and b-carotene were found in virtually all the strains tested, although at lower levels than those of lutein. Canthaxanthin and astaxanthin were present only in some strains and at very low levels, although a high astaxanthin content was found in C. zongiensis CCAP 211/14 (about 25 mg l 1 or 0.5 pg cell 1). Some of the strains considered in this work, such as C. fusca SAG 211-8b, C. citriforme, Muriellopsis sp., N. gelatinosum and C. zongiensis CCAP 211/14 are of potential practical interest on the basis of their high lutein level, the latter strain also exhibiting substantial amounts of astaxanthin. Among these, Muriellopsis sp. has been selected for further work focused on the production of lutein, since this alga also has the highest growth rate (0.170.23 h 1) and maximum standing cell density (up to 8 1010 cells l 1 culture). Fig. 1 shows the kinetics of growth and lutein accumulation in a batch culture of Muriellopsis sp. Both, lutein in the culture and lutein per cell increased with cell number during the exponential phase of growth, attaining a maximum of about 28 mg l 1 or 0.7 pg cell 1 in the early stationary phase. Violaxanthin and b-carotene followed the same trend as lutein, whereas maximal levels of

where x2 and x1 represent cell density values in terms of (cells ml 1) at times t2 and t1, respectively. For pigment analysis by HPLC, cells were ground in a mortar with alumina and the extracts prepared in pure acetone. Extracts were analyzed by the chromatographic method described by Mnguez-Mosquera et al. (1992), except that sam ples dissolved in acetone were centrifuged to discard particulate residues prior to injection into the liquid chromatograph, and that separation was performed on a Nova-Pak C18 column (3.9 150 , mm; 4-mm particle size; 60-A pore size) containing dimethyloctadecylsilyl bonded amorphous silica, protected with a guard cartridge (4 mm particle size, sealed with 2 mm lters). The pigments were eluted at a rate of 1 ml min 1, and were detected by measuring the absorbance at 360 700 nm,

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canthaxanthin and astaxanthin were recorded in the late stationary phase of growth (data not shown). Nitrogen availability affects carotenoid accumulation in some microalgae. Thus, nitrogen limitation increases the level of b-carotene in Dunaliella. In Haematococcus some authors have reported that astaxanthin accumulation is stimulated by nitrogen limitation, whereas others claim a requirement for nitrogen at not limiting concentrations (Borowitzka et al., 1991; Boussiba and Vonshak, 1991; Borowitzka, 1992). Nitrogen limitation resulted in a decreased level of lutein in cultures of Muriellopsis sp. As shown in Table 2, lutein in the culture was doubled when nitrate in the medium was increased from 10 to 20 mM, remaining practically constant at higher nitrate concentrations. On the other hand, the nature of the nitrogen source (NaNO3, NH4Cl or NH4NO3) did not inuence the lutein level in Muriellopsis sp. (data not shown). The data thus show that

high nitrogen concentrations favor accumulation of lutein in Muriellopsis sp., which might reect a need for continued synthesis of protein in order to support the massive accumulation of this carotenoid. The apparent discrepancy of our results with those of other authors for astaxanthin or b-carotene accumulation, aside from the fact that they refer to different carotenoids and microalgae, could be explained taking into account that irradiance values in this work are considerably higher. It is thus not surprising that lutein accumulation in Muriellopsis sp. at high irradiance may require an enhanced nitrogen supply. Since irradiance appears to induce increased levels of astaxanthin and b-carotene in Haematococcus and Dunaliella (Ben-Amotz et al., 1989; Kobayashi et al., 1992; Shaish et al., 1993; Fan et al., 1994), the effect of this factor on the levels of carotenoids has also been considered in Muriellopsis sp. (Table 3). Lutein in the culture was enhanced by about 40% as photon ux density

Table 1 Carotenoids in cultures of several strains of chlorophycean microalgaea Strain Carotenoid (mg l1 culture) Astaxanthin fusca, SAG 211-8bb fusca, SAG 211-8cb zongiensis, CCAP 211/14c zongiensis, CCAP 211/51d citriforme, SAG 62.80c proboscideum, var. dilatatum, SAG 217-2c C. proboscideum, var. gracile, SAG 217-3c M. aurantiaca, SAG 249-1d M. decolor, SAG 249-2d Muriellopsis sp.d N. gelatinosum, SAG B64.80c T. aplanosporum, SAG 91.80b T. intermedium, SAG 94.80d T. tetrasporum, SAG 98.80d Tetracystis sp.d C. C. C. C. C. C.
a

Biomass (g l1) b-Carotene 3.79 0.2 1.7 90.1 4.1 90.4 1.99 0.1 6.1 9 0.6 2.4 9 0.2 2.2 9 0.1 5.7 90.4 0.6 90.1 5.7 9 0.4 4.4 90.4 3.0 90.2 3.2 90.3 3.2 9 0.2 3.4 9 0.2 Lutein 23.2 91.9 12.5 9 1.0 18.9 91.5 6.3 90.3 38.0 93.6 15.691.3 11.0 90.7 17.2 9 1.5 5.0 90.3 29.8 91.8 29.8 9 2.8 3.4 90.2 13.3 9 1.2 14.0 91.1 13.7 91.1 Violaxanthin 3.0 90.2 1.6 90.1 1.4 90.1 1.0 90.1 7.9 9 0.6 1.7 90.2 2.1 90.2 4.2 90.3 nd 7.3 9 0.3 5.7 90.5 0.9 90.1 4.4 90.3 2.2 90.1 1.9 90.1 4.94 90.3 2.96 9 0.2 6.71 9 0.5 2.68 90.3 5.11 9 0.7 3.11 9 0.3 3.23 9 0.3 6.73 9 0.6 9.60 9 1.1 5.37 9 0.3 6.78 9 0.8 0.58 9 0.5 3.78 9 0.3 3.19 9 0.3 4.22 9 0.4

Canthaxanthin 0.5 9 0.02 nd 5.49 0.3 nd 1.89 0.2 nd nd nd 1.5 9 0.1 0.7 90.1 nd nd 2.990.2 nd nd

2.69 0.1 nd 24.89 2.1e nd 1.3 90.1 nd 1.79 0.1 0.19 0.01 nd 0.89 0.1 nd nd nd nd 0.19 0.02

Data correspond to maximum values attained in the culture, being the means9 S.D. of three independent measurements; nd, not detected. b Samples for biomass and carotenoids analysis were withdrawn at 1516 days of culture. c Samples for biomass and carotenoids analysis were withdrawn at 912 days of culture. d Samples for biomass and carotenoids analysis were withdrawn at 47 days of culture. e 9599% esteried.

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Fig. 1. Kinetics of growth and lutein accumulation in a batch culture of Muriellopsis sp. Symbols: , cell number; , lutein in the culture; , lutein per cell. Data shown represent mean values of at least three independent measurements, S.D. being lower than 10%.

increased from 184 to 460 mmol photon m 2 s 1, but decreased by about the same proportion at higher irradiance values. The lutein per cell followed a similar trend. Maximum standing cell density was similar at the different irradiances, but specic growth rate was doubled by increasing irradiance from 184 to 1725 mmol photon m 2 s 1. The highest levels of violaxanthin and bcarotene were also obtained at 460 mmol photon m 2 s 1, although the irradiance effect was less pronounced in these cases. Astaxanthin and canthaxanthin accumulation exhibited a different pattern, being maximal at the highest irradiance assayed (data not shown). An effect of pH on carotenoid accumulation has also been observed (Fig. 2). Maximum lutein in the culture (32.5 mg l 1) was obtained when cells were grown at pH 6.5, decreasing markedly at higher and lower pH values. However, when the data are alternatively expressed on a cell basis, maximum levels of lutein were attained at pH 6 and 9, being ve- to sevenfold higher than those

at pH 6.5. Similar effects were observed for violaxanthin and b-carotene (data not shown). Enhanced carotenoid accumulation at extreme pH values has also been reported for C. zongiensis and D. salina (Borowitzka, 1988). As shown in Table 4, lutein in the culture did not change signicantly by increasing NaCl concentration in the medium from 2 to 100 mM, but decreased at higher NaCl. This was a reection of
Table 2 Effect of nitrate concentration on lutein accumulation in cultures of Muriellopsis sp.a NaNO3 concentration (mM) 10 20 30 40 Lutein (mg l1 culture) 12.3 90.4 23.0 90.7 22.3 91.3 21.2 91.5

a Cells were grown at the indicated concentrations of NaNO3. Data are mean values 9 S.D. of three independent measurements in the early stationary phase of growth.

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Table 3 Effect of irradiance on lutein accumulation in Muriellopsis sp.a Photon ux density (mmol photon m2 s1) Lutein v (h1) Maximum standing cell density (1010 cells l1 culture)

(mg l1 culture) 184 460 1265 1725

a

(pg cell1) 0.4290.04 0.5190.05 0.3690.03 0.39 90.04 0.08 9 0.005 0.12 9 0.010 0.17 9 0.012 0.16 9 0.011 5.7 90.4 6.49 0.4 6.4 9 0.5 6.2 90.4

22.792.1 32.59 2.7 22.7 9 2.0 23.49 2.1

Cells were grown at the indicated irradiances. Data are mean values 9S.D. of three independent measurements in the early stationary phase of growth.

Fig. 2. Effect of pH on lutein accumulation and maximum standing cell density in cultures of Muriellopsis sp. Cells were grown at the indicated pH values, as described in Section 2. Lutein and cell number were determined in the early stationary phase of growth. Symbols: , maximum standing cell density; , lutein in the culture; , lutein per cell. Bars represent 9 S.D. of three independent measurements.

the effect of NaCl on cell growth, since both growth rate and maximum standing biomass were virtually unaffected by NaCl up to 100 mM, but decreased markedly at higher NaCl concentrations. Lutein per cell was virtually constant at the different NaCl concentrations assayed, as was the case for the other carotenoids (data not shown). Therefore, salinity does not seem to inuence

carotenoid accumulation in Muriellopsis sp., although it is a major factor inducing carotenogenesis in other chlorophycean algae, such as Haematococcus and Dunaliella (Borowitzka, 1988, 1992; Boussiba and Vonshak, 1991). The inuence of temperature on carotenoid level and growth of Muriellopsis sp. has also been examined. As shown in Fig. 3, the lutein per cell

J.A. Del Campo et al. / Journal of Biotechnology 76 (2000) 5159 Table 4 Effect of NaCl concentration on lutein content in cultures of Muriellopsis sp.a NaCl concentration (mM) 2 35 70 100 150 200
a

57

Lutein (mg l1 culture) 27.99 2.8 31.79 3.0 28.39 2.2 25.39 2.3 16.49 2.0 10.79 1.5

and b-carotene. This effect of temperature has also been reported for astaxanthin in Haematococcus and b-carotene in Dunaliella. It has been suggested that endogenously generated active oxygen is responsible for stimulation of carotenogenesis by high temperature in Haematococcus (Borowitzka, 1988; Tjahjono et al., 1994). Further research is required in order to prove whether this is also the case for Muriellopsis sp.

Cells were grown in the presence of NaCl added at the indicated concentrations. Data are mean values 9 S.D. of three independent measurements in the early stationary phase of growth.

4. Conclusions Muriellopsis sp., a lutein-rich strain, has been selected among a variety of chlorophycean microalgae as a potential source of this carotenoid, since it displays both the shortest doubling time and the highest maximum standing biomass of all the strains assayed. In general, optimal conditions for the growth of this strain also lead to maximal levels of lutein in the culture; accumulation of this

increased about sixfold as temperature was raised from 28 to 33C. Nevertheless, maximal lutein in the culture (about 30 mg l 1) was obtained at 28C, since maximum standing cell density was considerably higher at this temperature. Analogous results were obtained for violaxanthin

Fig. 3. Effect of temperature on lutein accumulation and maximum standing cell density in cultures of Muriellopsis sp. Cells were grown at the indicated temperatures and at a photon ux density of 950 mmol photon m 2 s 1. Lutein and cell number were determined in the early stationary phase of growth. Symbols: , maximum standing cell density; , lutein in the culture; , lutein per cell. Bars represent 9 S.D. of three independent measurements.

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J.A. Del Campo et al. / Journal of Biotechnology 76 (2000) 5159 Ben-Amotz, A., Shaish, A., Avron, M., 1989. Mode of action of the massively accumulated b-carotene of Dunaliella bardawil in protecting the alga against damage by excess irradiation. Plant Physiol. 91, 1040 1043. Benemann, J.R., 1992. Microalgae aquaculture feeds. J. Appl. Phycol. 4, 233 245. Borowitzka, M.A., 1988. Vitamins and ne chemicals from microalgae. In: Borowitzka, M.A., Borowitzka, L.J. (Eds.), Micro-algal Biotechnology. Cambridge University Press, Cambridge, pp. 153 196. Borowitzka, M.A., 1992. Comparing carotenogenesis in Dunaliella and Haematococcus: implications for commercial strategies. In: Villa, T.G., Abalde, J. (Eds.), Proles on Biotechnology. Servicio de Publicaciones, Universidad de Santiago, Santiago de Compostela, Spain, pp. 301 310. Borowitzka, M.A., Huisman, J.M., Osborn, A., 1991. Culture of the astaxanthin-producing green alga Haematococcus plu6ialis. I. Effects of nutrients on growth and cell type. J. Appl. Phycol. 3, 295 304. Bourrelly, P., 1990. Les algues deau douce. Initiation a la systematique, vol. 1. Les algues Vertes. Societe Nouvelle des editions Boubee, Paris. Boussiba, S., Vonshak, A., 1991. Astaxanthin accumulation in the green alga Haematococcus plu6ialis. Plant Cell Physiol. 32, 1077 1082. Broady, P.A., 1982. New records of chlorophycean micro-algae cultured from Antarctic terrestrial habitats. Nova Hedwigia 36, 445 479. Callegari, J.P., 1989. Feu vert pour les microalgues. Biofutur 76, 25 40. Chaumont, D., Thepenier, C., 1995. Carotenoid content in growing cells of Haematococcus plu6ialis during a sunlight cycle. J. Appl. Phycol. 7, 529 537. Eskling, M., Arvidson, P., Akerlund, H., 1997. The xanthophyll cycle, its regulation and components. Physiol. Plant. 100, 806 816. Fan, L., Vonshak, A., Boussiba, S., 1994. Effect of temperature and irradiance on growth of Haematococcus plu6ialis (Chlorophyceae). J. Phycol. 30, 829 833. Hagen, C., Braune, W., Vogel, K., Hader, P.P., 1993. Func tional aspects of secondary carotenoids in Haematococcus lacustris (Girod) Rostanski (Volvocales). V. Inuences on photomovement. Plant Cell Environ. 16, 991 995. Jacques, P.F., Chylack, L.T., McGandy, R.B., Hartz, S.C., 1988. Antioxidant status in persons with and without senile cataract. Arch. Ophthalmol. 106, 337 340. Johnson, E.A., Schroeder, W.A., 1995. Microbial carotenoids. In: Fiechter, A. (Ed.), Advances in Biochemical Engineering. Biotechnology 53, 119 178. Kobayashi, M., Kakizono, T., Nishio, N., Nagai, S., 1992. Effects of light intensity, light quality, and illumination cycle on astaxanthin formation in a green alga, Haematococcus plu6ialis. J. Ferment. Bioeng. 74, 61 63. Le Marchand, L., Hankin, J.H., Kolonel, L.N., Beecher, G.R., Wilkens, L.R., Zhao, L.P., 1993. Intake of specic carotenoids and lung cancer risk. Cancer Epidemiol. Biomarkers Prev. 2, 183 187.

carotenoid is enhanced in the early stationary phase. However, some stress conditions for growth, such as extreme pH and high temperatures, stimulate lutein cellular accumulation (pg cell 1). Therefore, a two-stage strategy could be suitable for achieving maximal lutein production, by rstly raising cells under optimal growth conditions until attaining maximum standing cell density and inducing thereafter carotenogenesis by imposing stress conditions. The performance of Muriellopsis sp. cultures outdoors is presently being veried in a 55-l capacity (2.2 m2 surface) tubular photobioreactor. A preliminary assessment of lutein production in spring (650750 mmol photon m 2 s 1) yielded values over 150 mg m 2 day 1, which are in the range of those reported for astaxanthin in Haematococcus and b-carotene in Dunaliella (BenAmotz and Mordhay, 1990; Borowitzka, 1992; Tjahjono et al., 1994; Chaumont and Thepenier, 1995). Muriellopsis sp. can be considered, therefore, a potential commercial source of lutein.

Acknowledgements The valuable technical assistance of M.J. Figueroa is very much appreciated. This work was supported by Comision Interministerial de Cien cia y Tecnologa, Spain (grants BIO94-0661 and BIO97-0577) and Plan Andaluz de Investigacion (group no. CVI 0131).

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