astaxanthin accumulation in the green alga haematococcus€¦ · astaxanthin, i.e., l2/n1 , pst/nl,...

Plant Cell Physiol. 32(7): 1077-1082 (1991)JSPP © 1991

Astaxanthin Accumulation in the Green Alga Haematococcus pluvialisl

Sammy Boussiba and Avigad Vonshak

Microalgal Biotechnology Laboratory, The Jacob Blaustein Institute for Desert Research,Ben-Gurion University of the Negev, Sede-Boker Campus, Israel 84990

Cells of the green microalga Haematococcus pluvialis were induced to accumulate theketocarotenoid pigment, astaxanthin. This induction was achieved by the application of thefollowing environmental conditions: light intensity (170//mol m~2s~'), phosphate starvationand salt stress (NaCl 0.8%). These conditions retarded cell growth as reflected by a decrease incell division rate, but led to an increase in astaxanthin content per cell. Accumulation of astaxan-thin required nitrogen and was associated with a change in the cell stage from biflagellatevegetative green cells to non-motile and large resting cells. It is suggested that environmental ornutritional stresses, which interfere with cell division, trigger the accumulation of astaxanthin.Indeed, when a specific inhibitor of cell division was applied, a massive accumulation of astaxan-thin occurred.

Key words: Astaxanthin — Cell division — Haematococcus — Light — Nitrogen — Phosphate —Salinity — Vinblastine.

The ketocarotenoid astaxanthin was first described inaquatic crustaceans as an oxidized form of ^-carotene,which gives the carapace of these animals its pinkish color(Czeczuga and Osorio 1989). It was later found that thispigment is very common in certain aquatic and landanimals. In some species of fish and birds it plays an im-portant role in coloration during the mating season (Czec-zuga and Osorio 1989). Astaxanthin is also found in somealgae such as Clamydomonas nivalis and Haematococcuspluvialis (Czygan 1970), Euglena rubida (Czeczuga 1974)and Acetabularia mediterranea. (Czeczuga 1986). It hasbeen suggested that nitrogen deficiency and high light in-tensity cause massive accumulation of this red pigment inthe green microalga H. pluvialis (Droop 1954, Goodwinand Jamikorn 1954, Czygan 1970). A different hypothesishas been put forward that the accumulation of astaxanthinin H. pluvialis is favored by agents which prevent cell divi-sion without impairing the ability of the alga to assimilatecarbon (Droop 1955). Another hypothesis suggests thatthe carbon-nitrogen balanace in the medium determines thedegree of carotene formation (Chodat 1938). The presentstudy was aimed at testing these hypotheses and at deter-mining the role of nutritional and environmental condi-

Abbreviation: DMSO, dimethylsulfoxide.1 Contribution No. 55 of The Microalgal BiotechnologyLaboratory.

tions involved in the induction and accumulation of thispigment.

Materials and Methods

Organism—Haematococcus pluvialis Flotow (Chloro-phyceae, order Volvocales) was obtained from the culturecollection of algae at the University of Gottingen.

Growth conditions—The algae were cultivated in amodified BGM medium (Stanier et al. 1971), which contain-ed 1.5 g liter"1 NaNO3 (Nl) or 0.15 g liter"1 NaNO3 (N2) in500 ml sterilized columns placed in a transparent plexiglasscirculating water bath. Water temperature was main-tained at 28°C. Light was supplied at photon flux densityof 85/flnolm~2s~' (LI), or 170/imolm~2s~' (L2). Con-tinuous aeration was provided by bubbling air containing1.5% CO2. Under these conditions the pH was main-tained between 6.8 and 7.0 Alga cultured under N l /L l(control) conditions remained green and motile until theyentered the stationary phase. Logarithmic cells from suchcultures were used for all experiments.

In some experiments NaCl was added to a final concen-tration of 0.8% (salt stress, Ss), and in others phosphatewas omitted from the growth medium (phosphate starva-tion, Pst).

Measurements of growth parameters—Cell numberwas determined with a Thomas blood cell counter. Chi

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1078 S. Boussiba and A. Vonshak

and astaxanthin were extracted with DMSO, as describedbelow. For Chi determination, cells were harvested by cen-trifugation, and the pellet was resuspended in 99.5%DMSO and homogenized. The mixture was then heatedfor lOmin at 70°C. If necessary, the procedure wasrepeated until a white pellet was obtained. The absorb-ance of the combined DSMO extracts was determined at673 nm, and Chi content was calculated according to Seelyet al. (1972). For astaxanthin determination, harvestedcells were first treated with a solution of 5% KOH in 30%methanol to destroy the Chi. The supernatant was discard-ed, and the remaining pellet was extracted with DMSO torecover the astaxanthin. This last step was repeated ifnecessary until the cell debris was totally white. The ab-sorbance of the combined extracts was determined at 492nm, and the amount of the pigment was calculated accord-ing to Davies (1976). Protein was determined according toLowry et la. (1951).

Other analyses—Ash-free dry weight and total lipidswere measured as previously described (Boussiba et al.1988). Carbohydrates were estimated according to the an-throne method (Hassid and Abraham 1957).

Results

Effect of light and nitrogen—Increasing light intensityto 170/imol m~2 s"1 (L2) or reducing the nitrogen contentin the medium to 0.15 g liter"1 (N2) reduced the growthrate of H.pluvialis (Fig. la). In both cases, cell divisionlevelled off by the fourth day of growth. When cells wereexposed to both modifications (L2/N2), growth was fur-ther inhibited.

At low light intensity (Ll/Nl and L1/N2) cell astaxan-thin content remained almost constant at 8pgcell~'(Fig. lb). When inducing conditions were imposed by ex-posing the cultures to high light intensity, a massive ac-cumulation of astaxanthin was observed. At 170/imolm~2 s"1 (L2) and 1.5 g liter"1 of NaNO3 (Nl) the astaxan-thin level reached a value of 65 pg cell"1 while under thesame light conditions but a lower nitrogen concentration(N2) this level was somewhat lower, 52 pg cell"1. Theonset and rate of accumulation of astaxanthin differed inthe two regimes: for L2/N1 the massive accumulationstarted only on day 5, while for L2/N2 the accumulationstarted on day 2 and took place at a much faster rate.

Effect of phosphate starvation—The influence of phos-phate supply in combination with nitrogen on astaxanthinaccumulation was investigated. H.pluvialis cells grownunder the Ll/Nl regime were harvested and resuspended ina phosphate-free medium containing either high (Nl) orlow (N2) concentrations of nitrogen (Fig. 2). Phosphate-deprived cells exposed to the high nitrogen concentrationdid not divide at all, whereas those grown under the lowlevel of nitrogen grew for two days and then stopped

2 4 6 8Time (days)

Fig. 1 Effect of light intensity and nitrogen level on growth (a)and accumulation of astaxanthin (b) in H.pluvialis. - • - L I /Nl , - o L1/N2, - A - L2/N1, - A - L2/N2. Relative cell number N /No is the cell number at any given time (N) devided by the initialcell number (No). Cells were cultivated under conditions describ-ed in Materials and Methods. Light and nitrogen content weremodified as follows: LI, 80^mol m" 2 s" ' ; L2,175 fimNl, 1.5 g liter"1 NaNO3; N2, 0.15 g liter"1 NaNO3.

- 2 , - 1 .

(Fig. 2a). Astaxanthin accumulation followed the op-posite pattern: being maximal in the phosphate-deprivedculture containing a high nitrogen level (Fig. 2b). Itshould be noted that the rate and extent of astaxanthin ac-cumulation per cell in phosphate-deprived cells was almostirrespective of light intensity (85 or llOfimol m~2 s~').

Effect of salt stress—Exposing the cells to salt stress bythe addition of 0.8% NaCl to the growth medium causedcomplete cessation of growth (Fig. 3a). Growth arrest wasaccompanied by a massive accumulation of astaxanthin,which reached 80 pg cell"' after 6 days in comparison with18 pg cell"1 in the control treatment (Fig. 3b).

Effect of cell division—To further evaluate the interac-tion between cell division, nutrient levels and astaxanthinaccumulation, the effect of a cell-division inhibitor,

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Fig. 2 Effect of phosphate starvation on growth (a) and ac-cumulation of astaxanthin (b) in H.pluvialis. - • - L l /Nl , - • -Pst/Ll/Nl, - a - Pst/Ll/N2. Growth conditions as in Fig. 1.For phosphate starvation cells were harvested, washed and resus-pended in a BG,, medium free of phosphate.

vinblastine, was studied. Cell division in cultures grown atLl /Nl regime and exposed to 2 or 5 n% ml"1 of vinblastinewas completely inhibited (Fig. 4a). This inhibition wasfollowed by an increase in the accumulation of astaxanthinin the resting cells (Fig. 4a). This inhibition was followedby an increase in the accumulation of astaxanthin in theresting cells (Fig. 4b).

Morphological changes—Under all the inductive con-ditions that led to the accumulation of astaxanthin adramatic change in cell morphology took place: from agreen small motile cell, 4 to 5 pm in diameter (Fig. 5a) to ared large resting cell, 15 to 20 ̂ m in diameter (Fig. 5b).

Effect of growth conditions on cell composition—Thechemical composition of H. pluvialis cells grown under thedifferent growth conditions described above is shown inTable 1. Protein content was less than that in the Ll /Nlregime in all the treatments tested. The reduction was

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(days)

Fig. 3 Effect of salt stress on growth (a) and accumulation ofastaxanthin (b) in H.pluvialis. - • - L l /Nl , -A- Ll /Nl , 0.8%NaCl. Growth conditions as in Fig. 1, NaCl was added at thebeginning of the experiment.

most pronounced in the nitrogen-deficient cells (53 and63% of the control in L2/N2 and L1/N2 treatments, respec-tively). Nitrogen deficiency also caused a considerable ac-cumulation of carbohydrates, the accumulation beingunaffected by light intensity: the carbohydrates amountedto 74 and 69% of dry weight under high and low light in-tensity, respectively, as compared with 39% in the cont-rol. In the regimes that triggered the accumulation ofastaxanthin, i.e., L2/N1, Pst/Nl, and Ss/Nl, celllipid con-tent increased by about 25-30% as compared with the con-trol treatment.

Discussion

H. pluvialis cultures exposed to different types of en-vironmental and nutritional stresses undergo changes in

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Fig. 4 Effect of the cell division inhibitor vinblastine on cellgrowth (a) and astaxanthin accumulation (b) in H. pluvialis.

Ll/Nl control, -O- Ll/Nl + vinblastine 2/igmT - A -

vinblastine 5 /ig ml '. Growth conditions as in Fig. 1, the inhibi-tor was added at the beginning of the experiment.

cell form and composition. The most pronounced changeis the accumulation of astaxanthin in non-motile restingcells. It has previously been suggested that nitrogen defi-ciency and high light intensity cause massive accumulationof this red pigment (Goodwin and Jamikorn 1954). It hasalso been proposed that the accumulation was triggered byan increase in the C/N ratio (Chodat 1938). The resultspresented in this work do not support these suggestions:the types of stress applied by us affected cell composition.It is, however, clear that these compositional changes arecorrelated with the type of stress rather than with the induc-tion of carotenogenesis. Contrary to previous suggestions(Czygan 1970, Droop 1954), our study shows that the syn-thesis of astaxanthin requires nitrogen, most likely reflec-ting the need for continuous synthesis of protein in orderto support the massive accumulation of the pigment. Thisis demonstrated in the phosphate-deprived culture grownwith a limiting concentration of nitrate (Pst/N2) (Fig. 2),although conditions triggering the accumulation of the pig-ment were imposed i.e., inhibition of cell division and con-tinuous light. The synthesis of the pigment in the treat-ment Pst/N2 resembles the pattern of the Ll/Nl (control),and was far below the treatment in which nitrogen was sup-plied in excess (Fig. 2b). Further support for this conclu-sion can be found in the comparison of the treatments L2/Nl and L2/N2 (Fig. lb): the combination of nitrogenlimitation and high light intensity caused indeed the highestrate of astaxanthin synthesis, until the synthesis stoppedwhen nitrogen was depleted from the medium on the fifthday of the growth. When nitrogen was supplied in ex-cess, the pigment synthesis proceeded at the same rate andreached a higher level (Fig. lb).

From the experimental data presented so far, wewould like to suggest that astaxanthin accumulation is in-duced whenever a disturbance in cell division is imposed.Thus a slow down of cell division is a prerequisite for the ac-cumulation of astaxanthin. This was demonstrated by

Table 1 Composition of Haematococcus pluvialis cells cultivated under different environmental growth conditions

Treatments

Ll/NlL1/N2L2/N1L2/N2Ss/Ll/NlPst/Ll/Nl

Proteins

26.416.620.014.020.020.0

Lipids

8.78.3

10.37.4

11.610.8

% of totalCarbohydrates

39.069.044.074.045.048.0

dry weightAsh

2.02.22.01.02.02.0

Astaxanthin

0.60.51.71.41.71.9

Chlorophyll

1.00.70.60.40.50.6

LI, 80/miol m~2 s"1; Nl, 1.5 g liter"1 NaNO3. L2,175 timo\ m"2 s~'; N2,0.15 g liter"1 NaNO3. Ss, 0.8% NaCl; Pst, no phosphate ad-dition. Samples were withdrown after 6-8 days of exposure to the indicated conditions. Cells were harvested, freez-dried and 10 mg ofthe dry powder was used for the determination. These results are an average of 3 different determinations with a standard error of±5%.

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Astaxanthin accumulation in Haematococcus pluvialis 1081

Fig. 5 Micrographs of H. pluvialis cells grown under control conditions (a) and phosphate starvation (b). The bar represents 5 ftm.

following the kinetics of astaxanthin accumulation inthe two inducing treatments L2/N1 and L2/N2. In thelatter treatment, accumulation started on the second day(Fig. lb), as did the slow down in cell growth (Fig. la),while in the former treatment the massive accumulationstarted on the fifth day, again in keeping with the slowdown in cell division.

Further support to this hypothesis can be obtainedfrom the phosphate starvation and salt stress experiments.In both treatments cells were initially grown under non-in-ducing (control) conditions, and only when a disturbancein cell division was imposed by phosphate starvation or saltstress, did a massive accumulation of pigment take place(Figs. 2 and 3).

More direct support for this hypothesis comes fromthe experiment with the cell division inhibitor: here again,cells grown under non-inducing conditions were induced toaccumulate a massive quantities of astaxanthin by inhibi-tion of cell division (Fig. 4).

At this stage the role of astaxanthin accumulation isnot clear. In other green algae, such as Dunaliella bar-dawil, the accumulated pigment ^-carotene in this case pro-tects the photosynthetic apparatus from high radiation(Ben-Amotz et al. 1989). This is not probably the case inHaematococcus for the following reasons:

1) The astaxanthin in H. pluvialis is localized in thecytoplasm and not in the chloroplasts (Santos and Mes-quita 1984), unlike ^-carotene in D. bardawil which islocated in the interthylakoid space of the chloroplast (Ben-

Amotz et al. 1989).2) Whereas "red" D. bardawil cells continue to grow,

evolve oxygen and divide (Ben-Amotz et al. 1989),H. pluvialis cells once exposed to conditions leading to theproduction of astaxanthin lose almost all their photosyn-thetic activity (data not shown) and change into a totallydifferent cell form, most likely a resting form (cyst).

3) Unlike D. bardawil, which is an obligate photo-autotroph, H. pluvialis can grow under heterotrophic con-ditions in the dark, utilizing acetate as the carbon source.If under these conditions cell growth is disturbed (nitrogenlimitation), the culture will turn red (Droop, 1955).

The data presented in this work describe aphenomenon, in which carotenogenesis in a photosyntheticcell is linked to developmental changes. The role of thepigment in the developmental process is still unclear, andthe question of whether those two processes are ultimatelylinked should be studied further.

We wish to thank Ms. Rachel Guy for helpful discussions,Ms. Rosa Milkis for technical help and Ms. Dorot Imber forediting the manuscript.

References

Ben-Amotz, A., Shaish, A. and Avron, M. (1989) Mode of ac-tion of the massively accumulated ^-carotene of Dunaliella bar-dawil in protecting the alga against damage by excess irradia-

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tion. Plant Physiol. 91: 1040-1043.Boussiba, S., Sandbank, E., Shelef, G., Cohen, Z., Vonshak, A.,

Ben-Amotz, A., Arad, S. and Richmond, A. (1988) Outdoorcultivation of the marine microalga Isochrysis galbana in openreactors. Aquaculture 72: 247-253.

Chodat, F. (1938) Etudes sur la genese des carotenoids. Arch.Sci. (Geneve) 20: 96.

Czeczuga, B. (1974) Carotenoids in Euglena rubida Mainx.Comp. Biochem. Physiol. 48B: 349-354.

Czeczuga, B. (1986) Characteristic carotenoids in some phytoben-thos species in the coastal area of the Adriatic Sea. Ada Soc.Bot. Pol. 55: 601-609.

Czeczuga, B. and Osorio, H. S. (1989) Investigations on carote-noids in lichens. XXI. Astaxanthin, the dominant carotenoid insome lichens from Uruguay. Israel J. Bot. 38: 115-120.

Czygan, F.-C. (1970) Blood-rain and blood-snow: nitrogen-defi-cient cells of Haematococcus pluvialis and Chlamydomonasnivalis. Arch. Mikrobiol. 74: 69-76.

Davies, B. H. (1976) Carotenoids. In Chemistry and Biochemis-try of Plant Pigments. 2nd Edition, Vol. 2. Edited by Good-win, T. W. pp. 38-165. Academic Press, London.

Droop, M. R. (1954) Conditions governing haematochrome for-

mation and loss in the alga Haematococcus pluvialis. Arch.Mikrobiol. 20: 391-397.

Droop, M. R. (1955) Carotenogenesis in Haematococcuspluvialis. Nature 175: 42.

Goodwin, W. and Jamikorn, M. (1954) Studies incarotenogenesis. II. Carotenoid synthesis in the algaHaematococcus pluvialis. Biochem. J. 57: 376-381.

Hassid, W. Z. and Abraham, S. (1957) Chemical procedures foranalysis of polysaccharides. Methods Enzymol. 3: 34-50.

Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J.(1951) Protein measurement with the Folin phenol reagent. J.Biol. Chem. 193: 265-275.

Santos, M. F. and Mesquita, J. F. (1984) Ultrastructural study ofHaematococcus lacustris (Girod.) Rostafinski (Volvocales). I.Some aspects of carotenogenesis. Cytologia 49: 215-228.

Seely, G. R., Duncan, M. J. and Widaver, W. E. (1972)Preparative and analytical extraction of pigments from brownalgae with dimethylsulfoxide. Mar. Biol. 12: 184-188.

Stanier, R. Y., Kunisawa, M. M. and Cohen-Bazire, G. (1971)Purification and properties of unicellular blue-green algae(order Chroococcales). Bacteriol. Rev. 35: 171-201.

(Received April 22, 1991; Accepted August 6, 1991)

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