Evolution of pigment composition in Chlorella vulgaris

Bioresource Technology 57 (1996) 157-163 Copyright © 1996 Elsevier Science Limited Printed in Great Britain. All rights reserved 0960-8524/96 $15.00 ELSEVIER

PII:S0960-8524(96)00058-2

E V O L U T I O N OF PIGMENT COMPOSITION IN C H L O R E L L A VULGARIS L. G o u v e i a , a V. V e l o s o , " A. R e i s , b H. F e r n a n d e s , b J. N o v a i s a & J. E m p i s a * "Laborat6rio de Engenharia Bioquimica, Instituto Superior T~cnico, 1096 Lisboa Codex, Portugal bDepartamento de Energias Renov6veis, Instituto Nacional de Engenharia e Tecnologia Industrial, 1699 Lisboa Codex, Portugal.

(Received 28 December 1995; revised version received 24 April 1996; accepted 29 April 1996) tural modifications of the various carotenoids ingested in food (Goodwin, 1951). Carotenoid composition in the Chlorophyceae is similar to that of higher plants, and includes a-carotene, fl-carotene, neoxanthin, lutein, violaxanthin, antheraxanthin and zeaxanthin, integrated in chloroplast lamellae and generally designated as primary carotenoids (Young, 1993). These are in contrast to secondary carotenoids, which may include equinenone, hydroxyequinenone, canthaxanthin and astaxanthin, as such or esterified to higher or lesser degrees (Burczyk, 1987; Young, 1993). The present authors have developed methodology for the production of carotenogenic Chlorella vulgar& biomass which they have demonstrated to be a useful natural encapsulated feed ingredient, capable of inducing colour in products such as eggs (Gouveia et al., 1996a) and trout flesh (Gouveia et al., 1996b). Detailed knowledge of carotenogenesis is therefore instrumental in determining harvest time in order to obtain a desirable hue in the finished product. Microalgal biosynthesis of carotenoids is species dependent and regulated by age, depending markedly on stress imposed by enviromental conditions, such as deviations from normal values of salinity, temperature, heavy metal concentration and, above all, nitrogen availability and increased light intensity (Japanese Patent, 1989). As may be seen from Fig. 1., the primary carotenoids fl-carotene and lutein/zeaxanthin, as well as the secondary carotenoids astaxanthin and cantaxanthin, are included in microalgal carotenogenic pathways (Santos & Mesquita, 1984), and the latter become more important as carotenogenic circumstances predominate. Whereas in some species, such as Dunaliella salina, fl-carotene is the predominant carotenoid (Borowitzka & Borowitzka, 1990), in others such as Haematococcus pluvialis, carotenogenic changes go all the way to the secondary astaxanthin and its esters (Grung et al., 1992; Maillard, 1993).

Abstract

The onset of carotenogenesis in Chlorella vulgaris and the change in nature and concentration of pigments with time was studied. The succession of pigments observed was interpreted in terms of relative efficiencies of carotenoid interconversion pathways, and this might be used to monitor the progress of the carotenogenic process. This work is relevant to the use of dry Chlorella biomass, as a naturally encapsulated form of a natural colouring ingredient, in animal feed. Copyright © 1996 Elsevier Science Ltd. Key words: Astaxanthin, carotenogenesis, canthaxanthin, Chlorella vulgaris, chlorophyll, carotenoid, lutein, pigment.

INTRODUCTION The production of valuable biochemicals from microalgae is based on the exploitation of their relatively efficient photosynthetic machinery, their biomass constituing a reservoir of natural substances of commercial value (Arad & Yaron, 1992). Some species have developed metabolic pathways leading to the accumulation of particular chemicals in high quantity; such as carotenoids to protect against oxidation under conditions of light stress, glycerol, saccharose or proline to protect from hypersaline water, exocellular polysaccharides to survive dessication and unsaturated fatty acids to induce changes in membrane viscosity against temperature variations and salinity (De la Noue et al., 1990). Despite the wide distribution of carotenoids, extensive to the animal kingdom, their de novo synthesis is confined to the plant world, in particular to fungi, algae and higher plants. Animals, in contrast, depend totally on dietary intake for their supply of carotenoids. They are capable only of limited struc*Author to whom correspondence should be addressed. 157

158

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L. Gouveia, V. Veloso, A. Reis, H. Fernandes, J. Novais, J. Empis

\

\

.\1

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~-CAROTENE ~ p , ~

EQUINENONE

ZEAXANTHIN/LUTEIN

ASTAXANTHIN ESTERS o

ASTAXANTHIN o

CANTHAXANTHIN o Hd y

"

-

/'~

RO ~

- -

Fig. 1. Biosynthetic pathways of carotenoid formation (adapted from Bar et al., 1993; Maillard, 1993). The qualitative as well as quantitative composition of the visible absorption spectrum is specific for the species and the status of carotenogenesis, and may be changed by altering certain culture parameters. The shape of the absorption spectrum will relate to the carotenoid composition, and because of the marked application dependency of the optimum absorption pattern, it is worth studying in detail. Carotenogenesis in Chlorella vulgaris will have a variable duration, depending upon the various stress conditions imposed and their intensity, but it leads invariably to this effect of colour change, and spectral characteristics may be used to indicate the best application-dependent stage at which the culture may be harvested, as will be discussed. In this work the onset of carotenogenesis in Chlorella vulgaris under nutrient deficiency, salinity stress and stronger light intensity, concomitant with cell dilution, was described and the variation of carotenoid concentrations was monitored, as a function of time. The results obtained are further analysed and interpreted in terms of possible and plausible carotenoid interconversion pathway efficiencies.

METHODS Microalgae and culture The microalga used was Chlorella vulgaris Beijerinck (INETI 58). The culture was grown in a medium

(Vonshak & Maske, 1982) that contained, in grams per litre: KNO3, 1.250; KH2PO4, 1.250; MgSO4.7H20, 1.000; CaCI2, 0.084; H3BO3, 0.111; FeSO4.7H20, 0.050; ZnSOa.7H20, 0.088; MnCIz.4H20, 0.014; MOO3, 0.007; CuSO4.5H20, 0.016; Co(NO3)z.6H20, 0.005; Fe.EDTA, 0,5; pH = 6.8. After growth until depletion of nitrogen source, cells were diluted with salt water to a transmitance of ca. 60% and maintained in the reactor for 22 days under high light intensity (38/~E/s/m 2, measured in a Li-Cor-1925A Underwater Quantum Sensor), and salinity stress (30 g/1 NaCI) and nitrogen starvation. Two replicates were grown, and samples were recovered daily and kept frozen at -18 ° C until analysis.

Analysis Approximately 10 ml acetone were added to 100 mg of Chlorella vulgaris dry biomass, and after vigorous homogenization (10000 rpm for 2 min), the mixture was centrifuged and the pellet was reextracted with further 10 ml portions of acetone until white. The acetone extracts were combined and the carotenoids transferred to diethyl ether by salting out (adding 10% NaC1 solution). After separation the carotenoid extracts were filtered under anhydrous NaeSO4 and evaporated until dry under vacuum. Absorption spectra of extracts were measured in a spectophotometer Hitachi 2000 reading between 380 and 700 nm. Reversed-phase analysis of total extracts was also performed on a HPLC (Perkin Elmer) with a kt-Boundapak C18 column and a detector UV/VIS Waters 481 (2 = 460 nm), with acetonitrile: methanol:water (65:35:2) as eluent. Methanol and acetonitrile were HPLC-grade reagents used without further purification other than filtration and degassing. The pigments were eluted over 30 min with a flow rate of 1 ml/min. The identification of the individual components was accomplished in the following way: preparative TLC systems were developed in such a way that each component was separated. For each sample system, TLC of acetone extracts described above was performed against that of a standard and compared with chromatographs of previously saponified material, in order to determine which components were saponifiable. The esters eluted as two bands which, under HPLC conditions, had Rt of 17.28 min and 19.81 min (Fig. 2, peaks 9 and 10). Individual components were scratched off the TLC plate, redissolved in acetone, filtered over anhydrous Na2SO4 and evaporated to dryness. For each isolated component Rt was measured in two different solvent systems against that of a standard. For the astaxanthin esters no standard samples were available. Therefore, bands equivalent to peaks 9 and 10 (Fig. 2) were individually scratched off, taken into acetone, evaporated to dryness and saponified

Pigments of Chlorella vulgaris

A

159

3

B

8

9

1"0

1"5

10

minutes

Fig. 2. Reversed-phase HPLC of green (A) and orange (B) cells of ChloreUa vulgaris and respective photo-micrographs ( x 1000). Peak identification: 1. Neoxanthin (Rt = 4.46 min); 2. Violaxanthin (Rt = 4.96 min); 3. Lutein ( R t = 6.06 min); 4. Chlorophyll b ( R t = 8.57 min); 5. Chlorophyll a (Rt = 11.12 rain); 6. fl-Carotene (Rt = 17.19 min); 7. Astaxanthin (Rt = 5.41 min); 8. Canthaxanthin (Rt = 6.74 min); 9, 10. Astaxanthin esters ( R t = 17.28 and 19.81 min). See Methods for details of the chromatographic conditions.

1.000

ABS

Q.O00 400

500

600

709

Fig. 3. Absorption spectra of total pigment extracts during carotenogenesis (t = 0, 3, 5, 7, 10, 16 and 22 days, respectively) and respective colours of the culture.

Pigments of Chlorella vulgaris with K O H / M e O H (1%) for 1.30 h under nitrogen. These saponified extracts proved to be indistinguishable by H P L C from an added astaxanthin standard. Freeze-dried samples were used for determination of lipid composition. Lipid extraction and transesterification was performed according to Cohen et al. (1988a) and the resulting methyl ester mixtures analysed in a Varian 3300 gas-liquid chromatograph equipped with FID. Separation was carried out with a 0.32 mm x 30 m fused silica capillary column (film: 0.32 /~m) Omegawax 320 (Supelco) with He as carrier at a flow rate of 1.5 ml/ min. The column temperature was 180°C. Injector temperature, detector temperature and split ratio were, respectively, 250°C, 260°C and 100:1. Heptadecanoic acid (Merck) was used as internal standard. Lipid extraction and separation of individual components by thin-layer chromatography was described in previous publications (Reis et al., 1990; Veloso et al., 1991). Other lipidic standards were supplied by Sigma. Individual bands were scraped off and transesterified as stated above.

161

5 4,5 4

[ ] b-carot

[ ] free astax [ ] canthax

2,5

~ - o 1,5

[ ] lutein

O

[ ] est ast

~-

0,5 0 0

3

5

7 10 16 22

Time (days)

Fig. 4. Sum of the different caroteniod masses (pigment/ dry weight) (mg/g) during carotenogenesis. be seen from Table 1, whereas total lipid content increased from 15-20 to 40-50% afdw (ash free dry weight). DISCUSSION

RESULTS The evolution of the absorption spectrum of the pigments during carotenogenesis is shown in Fig. 3. In this figure, the modification of the pattern of the visible absorption spectrum is shown with carotenogenesis. For this purpose, the shapes of the absorption curves obtained at sucessive intervals were superimposed. Because of this representation it is not possible to lend any quantitative meaning to the vertical coordinate, but a shift of the absorption maximum from green to yellow and red was noted. Monitoring the absorbance of a constant volume of microalgal culture against time, at a given wavelength, is a more traditional way of inspecting status evolution; it may be seen from Fig. 3 that a wavelength in the proximity of 2 = 470 nm should be used for maximum discrimination. It is evident from Fig. 2 that chlorophyll a concentration decreased while canthaxanthin and astaxanthin were appearing, as may be seen from the photomicrographs and from H P L C spectra of pigments extracted from both the initial green (A) and from the orange-coloured Chlorella vulgaris dominant in the final stages (B). The evolution of the carotenoid pigments during carotenogenesis is shown in Fig. 4, where the sum of total carotenoids masses during the experiment is shown. Postulating a constant extinction coefficient for all carotenoids at 2 = 470 nm t~1% ~z~lcm = 2109) a rough estimate may be obtained for the percentage of carotenoids accounted for to be no less than 15%. Analysis of fatty acid composition from algal lipids was performed, yielding for the carotenogenic alga a distribution which was slightly different from that found in green Chlorella vulgaris lipids, as may

The results show that for the Chlorella which has been examined, although /?-carotene is biosynthesized initially and is present together with chlorophyll a during the logarithmic growth phase, its concentration decreases when the carotenogenic pathways are activated. Accepted carotenogenic pathways show the possibilities of various oxidative transformations of /?-carotene up to canthaxanthin, and of the hydroxylative pathways towards zeaxanthin/lutein. Either of these compounds may be considered as precursors of the hydroxylated/oxidized astaxanthin, as may be seen in Fig. 1. Equinenone was not found in this system, which is taken to indicate that if at all present its oxidation to canthaxanthin is quick, whereas zeaxanthin/lutein are initially predominant, indicating that their oxidation to astaxanthin is slow. It is therefore plausible, given the relatively early accumulation of canthaxanthin, that both pathways operate, even if equinenone is unaccounted for. Canthaxanthin may also be hydroxylated to astaxTable 1. Fatty acid composition from green and orange Chlorella vulgaris lipids

Fatty acid

Green (%)

Orange (%)

14:0 16:0 16:1 16:4 18:0 18:1 18:2 18:3

3.7 21.9 5.9 6.8 2.0 18.9 15.2 20.7

0.6 26.2 1.4 0.7 4.7 47.0 6.7 7.8

L. Gouveia, V. Veloso, A. Reis, H. Fernandes, J. Novais, J. Empis

162

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o,8

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_11-----41

0'4 -r

should be taken to ensure that absorbance at 2 = 470 nm is maximized after the canthaxanthin to total astaxanthin ratio has become constant. Future work must necessarily contemplate the various possibilities of further increasing the value of maximum absorbance.

02

0 0

I

I

I

I

I

f

3

5

7

10

16

22

Time (days) Fig. 5. Ratio of canthaxanthin to total astaxanthin (free plus esterified) during carotenogenesis. anthin, in which case hydroxylation may, or may not, be subject to regulation. The evolution of the relative concentration of canthaxanthin and astaxanthin is therefore an important feature. In Fig. 5 a plot of relative concentrations of canthaxanthin versus total astaxanthin (free plus esterified) reveals this early dominance of canthaxanthin followed by a decrease to a constant factor of 0.5, relative to total astaxanthin, against carotenogenesis time. Astaxanthin esters meanwhile accumulate all the time. These species are initially formed slower than canthaxanthin or astaxanthin, but their concentration eventually grows to a level which equals that of free astaxanthin. Progressive esterification of astaxanthin to the various ester forms does not apparently affect the plausible regulation, which is hinted at by the constant relative concentrations shown in Fig. 5. Regarding fatty acid evolution, it is noteworthy that compositional modifications are not unexpected because of the larger size and higher total amount of lipids in carotenogenic species. In this respect it may be noted that the higher mean temperature during carotenogenesis and the importance of the lipid pool in the large-sized individuals is reflected by an overall increase of oleic relative to linoleic and linolenic components. The carotenogenic stage of a Chlorella culture may be adequately monitored in terms of the visible absorption spectrum, as suggested by Fig. 3, together with the determination of the canthaxanthin to astaxanthin ratio. In order to see this, two possible applications of Chlorella biomass must be contrasted. When this material is used as an ingredient for colouring egg yolk (Gouveia et al., 1996a) it is important to have maximum lutein/zeaxanthin, and this may be translated into actual process control by ensuring the above-mentioned ratio is larger than unity. When the application is as an ingredient in fish feed (Gouveia et al., 1996b), the relatively easy ester hydrolysis in fish gut and the nature of the final hue show that the algal biomass maintains its value as a pigment as carotenogenesis progresses, and as astaxanthin is better than canthaxanthin (Skrede et al., 1990) care

ACKNOWLEDGEMENTS The authors wish to acknowledge INETI and its permission to use local facilities for microalgal mass production, Faculdade de Ci6ncias (Departamento de Fisiologia Vegetal) for the microscopic photographs and Luisa Gouveia acknowledges a maintenance grant from JNICT (BD 1785/91-IF).

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