Factors responsible for astaxanthin formation in the Chlorophyte Haematococcus pluvialis

Bioresource Technology 55 (1996) 207-214 © 1996 Elsevier Science Limited Printed in Great Britain. All rights reserved 0960-8524/96 $15.00 ELSEVIER

0960-8524(95)00002-8

FACTORS RESPONSIBLE FOR A S T A X A N T H I N F O R M A T I O N IN THE C H L O R O P H Y T E HAEMATOCOCCUS PLUVIALIS Mark Harker,* Alex J. Tsavalos & Andrew J. Young:~ School of Biological and Earth Sciences, John Moores University, Byrom Street, Liverpool L3 3AF, UK

(Received 17 October 1995; revised version received 5 December 1995; accepted 9 December 1995)

Abstract The accumulation of high levels of the ketocarotenoid astaxanthin by the green freshwater microalga Haematococcus pluvialis in response to extreme environmental conditions is a well recorded phenomenon. However, there is still considerable debate regarding the nature and possible interactions of the abiotic factors that are thought to be primarily responsible for stimulating the synthesis of this carotenoid. In this study H. pluvialis was exposed to a number of different nutritional and physical parameters to determine the effect each exerted on stimulating astaxanthin formation in the alga. When H. pluvialis was cultivated in media deficient in nitrogen, algal growth was limited severely and astaxanthin synthesis greatly stimulated. Similar stimulatory effects on carotenoid synthesis were observed in the presence of elevated levels of ferrous iron and, especially, when the alga was transferred into saline media. In both cases algal growth was once again severely limited. However, the single most important factor in terms of carotenogenesis was to subject the alga to high photon-flux densities. In contrast to these effects, transfer of the alga to phosphate-limiting conditions increased the rate of astaxanthin synthesis but, importantly, algal growth was not inhibited greatly. The extent to which each parameter tested was able to stimulate the formation of astaxanthin in the alga varied and the overall effectiveness of each treatment in promoting astaxanthin formation in the alga is discussed. Copyright © 1996 Elsevier Science Ltd.

INTRODUCTION

When exposed to extreme environmental conditions the Chlorophyte Haematococcus pluvialis accumulates large quantities of the ketocarotenoid astaxanthin ((3S,3'S)-3,3'-dihydroxy-fl,fl-carotene4,4'-dione). Under optimal growth conditions vegetative cells of the alga persist and the alga possesses carotenoids normally found in the Chlorophyta and in the chloroplasts of higher plants, namely fl-carotene, lutein, violaxanthin, neoxanthin and zeaxanthin (often referred to collectively as primary carotenoids). However, when the alga is exposed to growth-limiting conditions the vegetative cells begin to synthesise astaxanthin, at the same time undergoing changes in cell morphology resulting in the formation of large red aplanospores resistant to the prevailing extreme environmental conditions. The formation of astaxanthin in H. pluvialis is associated with large changes in the morphology, physiology and photosynthetic characteristics of the alga. These changes include the loss of two posterior flagella, rendering the aplanospores immobile (Elliot, 1934), the accumulation of large lipid bodies in the protoplast which contain the astaxanthin (Lang, 1968), the presence of a thick sporopollenin cell wall resistant to oxidative degradation (Burczyk, 1987) and lowered photosynthetic rates (Hagen et al., 1992). Other ketocarotenoids generally reported to be present in H. pluvialis under extreme environmental conditions include canthaxanthin, echinenone, adonirubin and //-carotene, although these together generally represent only a few percent of total carotenoid in fully encysted cells (Grung et al., 1992; Tsavalos et al., 1992). Those carotenoids possessing hydroxy groups (e.g. astaxanthin) usually occur in algae such as Haematococcus spp., Oocystis spp., etc. as a complex mixture of fatty acid mono- and bis-esters. These carotenoid

Key words: Astaxanthin, carotenoid, Haematococcus.

*Present address: Dept. of Genetics, The Hebrew University of Jerusalem, Givat Ran Campus, Jerusalem 91904, Israel. ~tAuthor to whom correspondence should be addressed. 207

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acyl esters can account for up to 98% of total carotenoid in this particular algal strain. The exact ratio of mono" bis-esters appears to be dependent upon the age of the culture (Harker et al., 1996). The function of the ketocarotenoids present in lipid globules outside the chloroplast is unknown. In studies involving the alga Eremosphaera viridis, astaxanthin was detected in the light-harvesting protein of PSI, suggesting that this carotenoid provided protection of the photosynthetic pigment protein complex against photosensitised damage (Vechtel et al., 1992), involving a mechanism similar to that observed in the functioning of primary carotenoids (Siefermann-Harms, 1987; Young, 1993). In aplanospores of H. pluvialis the primary function of astaxanthin is thought to be as a 'passive' photoprotectant (i.e. a filter), simply, but effectively, reducing the amount of light available to the lightharvesting pigment protein complexes of PSII, minimising the risk of photoinhibition (Yong & Lee, 1991; Hagen et al., 1994). The esterification of the relatively polar, hydroxyl-containing astaxanthin with fatty acids may increase the photoprotective efficiency of the astaxanthin by improving the solubility of the carotenoid, localising the chromophore within cytoplasmic globules in the protoplast of the cells (Bidigare et al., 1993). Further protective activities of the extra-chloroplastic astaxanthin in H. pluvialis may include providing a physico-chemical protective barrier, preventing photodynamic free-radical-mediated damage within the cell. As astaxanthin surrounds the nucleus initially before filling the entire protoplast, it is possible that this involves specific protection of the cells genetic material. Astaxanthin (a more effective quencher of singlet oxygen than fl-carotene) has the potential to stabilise membrane systems and act as a buffer for oxidative reactions, as well as quenching radical reactions, which could ultimately lead to the death of the cell (Hagen et al., 1993). Recently, there has been considerable experimental investigation into the conditions responsible for promoting astaxanthin formation in H. pluvialis. This alga (along with the yeast Phaffia rhodozyma, for example) is being assessed as a potential commercial source of a natural form of astaxanthin for use in the aquaculture industry as an alternative to the existing synthetic product. However, much of the published data on the cultivation of this alga leading to the accumulation of astaxanthin is contradictory, with different studies reaching separate conclusions as to which factors induce astaxanthin formation in this alga. These contradictions may, in part, be accounted for by the experimental design adopted in many of the published experiments. The present work evaluates the effects of nitrate, phosphate, ferrous iron, salt and light intensity on the formation of astaxanthin in H. pluvialis with each parameter tested independently of each other, enabling the

contribution of each to astaxanthin formation on the alga to be determined.

METHODS Organism and growth medium Haematococcus pluvialis (Flotow) 34/7 was obtained from the Culture Collection of Algae and Protozoa, Windermere, UK. The alga was cultivated in Bold's Basal Medium (Nichols & Bold, 1964), modified to pH 7.0. Nitrate, Phosphate and iron The nitrate concentration of the media was varied by altering the level of NaNO3 in the culture medium. The nitrate concentrations investigated were 0.0, 0.75, 1.50, 3.00 and 6.00 mM. The phosphate concentration of the culture medium was varied by altering the levels of K2HPO4 and KH2PO4 in the media. The phosphate concentrations investigated were 0.85, 1.70 and 3.40 mM. Control levels of nitrate and phosphate were those of the BBM medium (3.00 and 1.70 raM, respectively). Three 250 ml Erlenmeyer conical flasks containing 50 ml BBM modified to the appropriate nitrate or phosphate concentration were inoculated with 5 ml of algal suspension. The flasks were kept on an orbital shaker at 80 rpm and the cultures maintained at 22°C. Continuous light was supplied by cool white fluorescent tubes at an irradiance of 35/~mol photons m -2 s-1 (PAR). A known volume of iron solution (supplied as FeSO4.7H20) was filtered aseptically (2 pm, Millipore) into 250 ml Erlenmeyer conical flasks containing 50 ml BBM to avoid possible contamination of the cultures. The iron concentrations investigated were 18.0 (the level in BBM used as the control), 36.0 and 72.0 /~M. Three flasks of each iron concentration were prepared and inoculated with 5 ml of H. pluvialis suspension. The flasks were incubated as described above. For all three experiments an aliquot of algal suspension was taken from each flask every 7 days for growth and pigment analysis by reversed-phase HPLC. Salt The alga was initially grown in a 5 1 bioreactor to produce sufficient algal biomass. Known volumes of algal suspension (50 ml) were then transferred aseptically into 250 ml Erlenmeyer conical flasks and cultured as described above. The concentrations of KCI and NaC1 used in the experiments were 0.0, 40.0, 70.0 and 100.0 mM. Samples were taken and analysed as described in the previous experiments. Light Light intensity was investigated as an environmental factor which was expected to have a pronounced effect on astaxanthin formation in H. pluvialis. The alga was initially grown in a 5 1 bioreactor to pro-

Factors causing astaxanthin formation in chlorophytes 3.0

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Fig. 1. Effect of nitrate concentration on (a) cell number and (b) astaxanthin formation in cells of H. pluvialis. Symbols: • 0.0; o 0.75; • 1.5; [] 3.0; • 6.0 mM nitrate (S.E. + 6.2%; n = 3 ) . duce sufficient algal biomass. Known volumes of algal suspension (50 ml) were then transferred into 250 ml Erlenmeyer conical flasks and the cultures exposed to three different light intensifies [2, 37 and 89 #mol photons m - 2 S -1 (PAR)]. Cell numbers and carotenoid contents were determined over a period of 30 days as described above. Analytical methods Algal cell growth was determined by cell number counting with an improved Neubauer haemocytometer. Total pigments (chlorophyll and carotenoid) were extracted, using a tissue homogeniser (Mickle Engineering Co. Ltd, UK), in 100% acetone. Samples were then filtered, dried and stored under a nitrogen atmosphere at -20°C until subsequent HPLC analysis. Individual carotenoids were quantified using reversed-phase HPLC as described previously (Tsavalos et al., 1992). Statistical analysis The data were subjected to statistical analysis to determine the standard errors of the means. Significant differences between the treatments were calculated using the data obtained on the final day of the experiments. These were determined by calculating the least significant difference (LSD) of the means. The LSD is represented as a single bar on each of the graphs, thus any points on the graph on the final day of the experiment which are separated by a distance which is greater than the length of the bar are considered to be significantly different. RESULTS AND DISCUSSION The results indicate clearly that many nutritional and environmental parameters are potential inducers of astaxanthin formation in cells of H. pluvialis. When cultivated in fresh BBM the alga can remain in a green, vegetative, state for a considerable period of time and it is only when the cultures age and nutrients such as nitrate are depleted that growth becomes limited, the cells encyst and accu-

mulate astaxanthin. Figure l(a) and (b) shows the effect when vegetative cells of H. pluvialis were cultivated in a range of nitrate concentrations. By varying the concentration of nitrate in the medium, the alga could be manipulated with respect to both growth and astaxanthin formation; at lower nitrate concentrations algal growth was limited severely and high levels of astaxanthin accumulated in the surviving cells. Levels of total carotenoid (of which astaxanthin represents >95%) in excess of 300 pg per cell were synthesised in 30 days. These results are in agreement with Spencer (1989) and Goodwin & Jamikorn (1954) in that nitrogen-deficient conditions induce astaxanthin formation in cells of /4. pluvialis. Indeed, astaxanthin accumulation can be independent of nitrogen concentration completely (Droop, 1954). In contrast, Boussiba et al. (1992) reported that nitrogen is an important requirement for astaxanthin synthesis in the alga, a view contrary to studies which indicate that a high ratio of C:N will greatly stimulate carotenogenesis and encystment in Haematococcus (Kakizono et al., 1992). Phosphate starvation has been reported previously to act as a trigger for the accumulation of astaxanthin (Boussiba & Vonshak, 1991). However, other authors have suggested that high phosphate concentrations stimulate the production of astaxanthin within algal cells (Borowitzka et al., 1991). Although variable, the overall effect of phosphatelimiting conditions [as shown in Fig. 2(a) and (b)] on H. pluvialis was similar to the effects observed in nitrogen-limited cultures; i.e. phosphate starvation stimulated astaxanthin synthesis in this alga. It is, however, important to note that a reduction in phosphate levels did not inhibit growth as severely as nitrogen starvation in this alga and the levels of astaxanthin produced per unit volume of culture might be considerably higher as a result. The results obtained in the present study support further the supposition that it is exposure to low, and not elevated, phosphate levels which induces astaxanthin formation in cells of H. pluvialis. The concentration of ferrous iron present in the culture medium exerted an effect on astaxanthin

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M. Harker, A. J. Tsavalos, A. J. Young 200

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Fig. 2. Effect of phosphate concentration on (a) cell number and (b) astaxanthin formation in cells of H. pluvialis. Symbols: • 0.85; o 1.7; • 3.4 mM phosphate (S.E. + 5.8%; n = 3). accumulation only at relatively high concentrations, as indicated in Fig. 3(a) and (b). Growth in the presence of low levels of ferrous iron produced little change in terms of either cell growth or astaxanthin formation from the control levels. Algal growth was inhibited at the highest iron concentration (72.0/ZM). These cells did, however, accumulate astaxanthin, although levels of total carotenoid were much lower than those obtained by manipulation of the nitrate and/or phosphate levels in the growth medium. Previous studies have also reported an elevation in astaxanthin formation caused by increased iron concentrations (Kobayashi et al., 1991). The addition of EDTA-chelated FeC13.6H20 (a form of iron which does not undergo Fenton chemistry) did not produce any significant differences in astaxanthin formation when added at three different concentrations (Borowitzka et al., 1991). The ferrous form of Fe in particular is known to give rise to free radical formation (especially hydroxyl radicals, HO') via Fenton chemistry. Radical initiation of cellular processes via signal cascade systems is thought to be an integral part of many biological systems and it has been suggested that free radicals may play a role in the accumulation of fl-carotene in Dunaliella (BenAmotz & Avron, 1983) and astaxanthin in the yeast P rhodozyma (Schroeder & Johnson, 1995). Kobayashi et al. (1993) reported that increased astaxanthin formation caused by Fe 2÷ was inhibited by potas3.0

sium iodide which scavenges HO., suggesting the HO" formed by an iron-catalysed Fenton reaction is required for enhanced astaxanthin biosynthesis in H. pluvialis. The same workers also reported that four active oxygen species (namely 102, H202, peroxyl radical and the superoxide anion radical) also stimulated the formation of astaxanthin in this alga. However, in the astaxanthin-accumulating yeast, P rhodozyma, only exposure to exogenous 102 (but not peroxyl radicals o r H 2 0 2 ) stimulated carotenoid synthesis (Schroeder & Johnson, 1995). The effects of a number of other metals (Cu 2+, M n 2+ and Cd2+), as well as Fe 2+, on carotenogenesis in Haematococcus were also studied. All except Cu 2+ increased carotenoid content on a cellular basis in the alga but algal growth was severely inhibited (Table 1). Of the metals tested, only the addition of Fe 2+ (the only one of these metals to sustain Fenton chemistry) to the growth medium resulted in an increase in carotenoid levels on a unit volume basis (13% greater than the control) but all treatments increased carotenoid content per algal cell. The addition of 0.5 ]2M H 2 0 2 also stimulated astaxanthin synthesis on a per cell basis (Table 1), although exposure to higher levels, or for longer than 9-10 days, severely inhibited growth of the alga (data not shown). Taken together with the data shown here [Fig. 3(a) and (b)] this may support further the hypothesis that oxidative stress may be one mechanism leading to 50

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Fig. 3. Effect of iron concentration on (a) cell number and (b) astaxanthin formation in cells of H. pluvialis. Symbols: • 18; o 36; • 72 #M Fe (S.E. ___4.9%; n = 3).

Factors causing astaxanthin formation in chlorophytes

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Table 1. Effects of metals and H202 on the growth and carotenoid content of H. pluvialis after 30 days and 9 days, respectively. Values are the means of at least three replicates and are shown as a percentage of the values obtained in the control (BBM) medium. The concentrations employed were determined by a series of preliminary experiments. The carotenoid composition (as determined by HPLC analysis) of the alga was unchanged by all treatments compared to the control, and astaxanthin esters accounted for 95% of total carotenoid in all treatments

Treatment

Cell number/ml (% of control)

Carotenoid/ml (% of control)

Carotenoid/cell (% of control)

88.6 62.8 48.5 86.1

113.2 84.8 64.2 106.5

127.6 136.2 134.5 123.0

Fe (0.4 mM) Mn (0.4 mM) Cd (2/~M) H 2 O 2 (1 #M)

astaxanthin formation in the alga, although levels of astaxanthin are low compared to those obtained by exposure to low levels of key nutrients. Clearly further studies are needed to establish the effects of selected oxygen radicals species on the levels of certain key enzymes involved in the early stages of carotenoid synthesis (e.g. phytoene synthase, phytoene desaturase). Figure 4 and Fig. 5 show that increases in the salinity of the culture medium (by addition of NaC1 and KC1, respectively) also resulted in the initiation of astaxanthin formation in the alga, with the highest levels of astaxanthin being formed at 100 mM NaC1.

However, such increases in salinity were accompanied by high rates of cell mortality, with those few cells which remained viable under such conditions accumulating very high levels of astaxanthin (500 pg carotenoid per cell) in comparison to many of the other treatments used in this study. It is not known whether the increased capacity some cells possess for producing high levels of astaxanthin is one of the reasons why these particular cells are able to survive under such severe culture conditions. The addition of KC1 to the algal cultures, even at low concentrations (i.e. 40 raM), resulted in relatively high rates of cell mortality [Fig. 5(a)]. The results show that the 600

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Fig. 4. Effect of NaC1 concentration on (a) cell number and (b) astaxanthin formation in cells of H. pluvialis. Symbols: •

0.0; © 40; • 70; [] 100 mM NaC1 (S.E. + 5.7%; n = 3)

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Fig. 5. Effect of KC1 concentration on (a) cell number and (b) astaxanthin formation in cells of H. pluvialis. Symbols: • 0.0; © 40; • 70; [] 100 mM KC1 (S.E. + 6.1%; n = 3).

M. Harker, A. J. Tsavalos, A. J. Young

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addition of potassium to the cultures, even at low concentrations, produced a phytotoxic effect. The strong phytotoxic effects of the chloride ions from KCI can be discarded, as such an effect was not observed when NaC1 was added to the cultures and it is likely that the K + ions are toxic. It has been suggested that algal cells do not possess an efficient extrusion mechanism for K + as they have for Na + and CI- (Pick et al., 1986). The exposure of 14. pluvial& cells to increased salinity has been reported previously to induce astaxanthin formation in the alga (Borowitzka et al., 1991; Boussiba & Vonshak, 1991; Spencer, 1989). All the available data agree that exposure to increased salinity induces astaxanthin formation in the alga. Although NaCI was used to induce astaxanthin formation in H. pluvialis, none of those studies presented any data, or inferred, that they had tested the alga in a range of salt concentrations to achieve maximum rates of astaxanthin formation. For example, the optimum concentration of NaCI that gave maximum astaxanthin levels per unit volume of culture was identified as 40 mM (data not shown), while the highest level of astaxanthin per algal cell was obtained at 100 mM NaC1, a salt concentration at which high rates of cell death were observed. The optimum NaC1 concentration obtained in this study is similar to the concentration used by Spencer (1989), who used 50 mM NaCI to induce astaxanthin formation in H. pluvialis. In contrast, KCI had a detrimental effect on algal growth and hence astaxanthin formation per unit volume of culture medium at all concentrations. Figure 6(a) and (b) shows that the light intensity the H. pluvialis cultures were exposed to had a significant effect on the level of astaxanthin accumulated in the cells. High light intensities caused relatively large quantities of astaxanthin to be accumulated in the cells of H. pluvialis. Even though exposure to high light intensities resulted in high rates of cell mortality, the cells which did survive contained large quantities of astaxanthin. At lower light intensities the amount of astaxanthin accumulated was in comparison very low but the survival rates of the alga 1.6

were increased significantly. Further studies involving the use of Response Surface Methodology (Harker et al., 1995) have indicated that the optimum light intensity for the induction of astaxanthin synthesis in the alga is in the range 1550-1650 #tool photons m -2 s-1 (PAR), which is considerably higher than any of the light intensities investigated in the present study. However, these results, along with those of other groups, illustrate that light is one of the most important factors responsible for astaxanthin formation in H. pluvialis (Boussiba et al., 1992; Donkin, 1976; Kobayashi et al., 1992). Contrary to a previous report (Goodwin & Jamikorn, 1954), astaxanthin formation in the absence of light can occur, albeit at a much reduced rate. Grown on an acetatebased BBM, dark-grown cultures of H. pluvial& have been shown to accumulate traces ( < 0 . 1 % of algal dry weight compared to levels >2.5% in the light) of astaxanthin (mainly as mono-esters). Astaxanthin esters may, however, account for up to 98% of all carotenoid within the aplanospores produced in the dark-grown cultures. Rates of growth were significantly lower in the dark than in the same, acetate-based, medium in the light (see also Droop, 1955). Astaxanthin production has been shown to be enhanced when the alga is grown under blue light as opposed to white or red light (Kobayashi et al., 1992). The same authors also reported that continuous illumination rather than light/dark illumination cycles are more favourable for astaxanthin formation. All the various parameters investigated in the present study stimulated the formation of astaxanthin in cells of H. pluvialis, with each parameter exerting a differential effect on the rate of astaxanthin synthesised in the alga. This is clearly seen in Table 2, in which the effects of the different treatments used in the present study are shown in terms of (i) rate of algal growth and (ii) rate of astaxanthin accumulation per cell. All treatments resulted in astaxanthin synthesis in H. pluvial&, but few treatments combined a high rate of astaxanthin synthesis in the alga with an increase in growth and many, in fact, resulted in a decrease in growth rate (e.g. expo500

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Fig. 6. Effect of light intensity on (a) cell number and (b) astaxanthin formation in cells of H. pluvialis. Symbols: • 2.0; © 37.0; • 89/~mol photons m -2 s i (PAR) (S.E. _+ 5.1%; n = 3).

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Table 2. Effects of alterations to the medium composition and irradiance on the rate of growth and rate of astaxanthin accumulation in H. pluvialis over 30 days

Treatment

Mean rate of growth ( x 103 cell/ml/day)

Mean rate of astaxanthin accumulation (pg/cell/day)

(a) Nitrate (mM) 0"00 0"75 1"50 3'00 6'00 (b) Phosphate (mM) 0'75 1"70 3"40 Iron (/~M) 18"0 36"0 72'0 (d) NaCl (mM) 0"0 40'0 70"0 100"0 (e) KCI (mM) 0"0 40-0 70"0 100"0 (f) Irradiance (/~moi photons m-2s l) 2 37 89

sure to high salinities). Whilst it is important to identify those conditions that promote astaxanthin synthesis within the alga, it is equally important to be aware of the potentially deleterious effects of such treatments on algal growth and hence carotenoid production per unit volume of culture. It can be concluded that any factor which interferes with certain cellular processes within the alga (and hence limits growth or photosynthesis) can act as a trigger for the process of encystment and astaxanthin formation. It would appear that microaigae synthesise and store astaxanthin with environmental and nutritional factors regulating this function. No one has yet elucidated the mechanism by which stress conditions promote the production of lipids in microalgae. However, a role for abscisic acid and other higher-plant hormones, such as ethylene, has been suggested (Maillard et al., 1993). Moreover, it has not yet been determined whether the enhanced lipid production is the result of catabolism of preformed cellular carbohydrates and/or proteins or the result of de novo synthesis by regulation (or deregulation) of the photosynthetic pathway. One suggestion proposed for the accumulation of lipids under adverse conditions is that mitosis becomes inhibited (Suen et al., 1987). The cell cycle is thought to be interrupted at a position responsible primarily for the synthesis of lipids. As mitosis is impeded, lipid content increases, which leads to the formation of cytosoline bodies.

Fully-bleached (i.e. 'ghost' cells containing no chlorophyll or carotenoid) cells were observed regularly in many of the cultures throughout this study. The number of these 'ghost' cells increased considerably when the alga was exposed to increasingly adverse environmental conditions; i.e. high photonflux densities and high salinity. Such bleaching is thought to occur as a result of metabolic imbalances, combined with photo-oxidative events within the cells (Sandgren, 1983). The results presented in the current investigation indicate that various environmental and nutritional factors invoke a differential response within the alga. The degree of this response is dependent upon the individual factor (stress) to which the alga is exposed, suggesting that the alga is able to perceive the various stresses differently. In conclusion, a number of factors relating to the cultivation of the microalga Haematococcus pluvialis can lead to the synthesis of the carotenoid astaxanthin and have been identified. Reductions in nitrate and phosphate and the addition of NaC1 to the culture medium are most effective, especially when combined with exposure to high photon-flux densities (Harker et al., 1995). In the majority of cases this is accompanied by encystment of the alga, particularly where the synthesis of the carotenoid is rather prolonged, or even extensive cell-death. Such effects can be minimised by exposure to very high photon-flux densities (Harker et al., 1995) or, for

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M. Harker, A. J. Tsavalos, A. J. Young

example, by addition of NaC1 in stages to achieve the final desired concentration.

ACKNOWLEDGEMENTS This work was supported by a John Moores University Research Grant and by the E u r o p e a n Agriculture and Fisheries Research Programme (AIR2 CT94-1283).

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