JOURNAL OF FERMENTATION AND BIOENGINEERING

Vol. 71, No. 5, 335-339. 1991

Astaxanthin Production by a Green Alga, Haematococcus pluvialis

Accompanied with Morphological Changes in Acetate Media
MAKIO KOBAYASHI, T O S H I H I D E KAKIZONO, AND SHIRO NAGAI*
Department of Fermentation Technology, Faculty of Engineering, Hiroshima University, Kagamiyama,
Higashi-Hiroshima 724, Japan
Received 24 October 1990/Accepted 6 February 1991

Two acetate containing media were developed for astaxanthin production by a green unicellular alga,
Haematococcus pluvialis. The basal medium, a vegetative growth medium facilitated the algal cell growth,
whereas the modified medium was likely to induce morphological changes with the formation of large cysts
and bleached cells which seemed to consequently enhance the carotenoid biosynthesis. In the two-stage culture,
the injection of ferrous ion with acetate into the basal medium on the fourth day, was greatly stimulative for
both the algal cell growth and the astaxanthin formation at a high light intensity. In addition, carotenoid
precursors, mevalonate and pyruvate were effective on the carotenoid formation in the modified medium.
Pyruvate was an especially good carbon source both for the algal cell growth and the carotenoid synthesis.

Astaxanthin (3,3'-dihydroxy-/3,/3'-carotene-4,4'-dione) has Media composition and growth conditions The

attained commercial interest as a pigmentation source basal medium (pH 6.8) was prepared based on that of
both for salmonid aquaculture and egg yolk in poultry in- Euglena (9): sodium acetate, 14.6mM; L-asparagine,
dustry (1). In addition, it has been shown that this carote- 2.7 mM; yeast extract, 2.0 g/l; MgC12.6H20, 0.985 mM;
noid would possess a higher antioxidant activity than /3- FeSO4.7H20, 0.036mM and CaC1E.2H20, 0.135 mM us-
carotene (2) and a-tocopherol (Miki, W., Ninth Int. Symp. ing deionized water. A ten ml portion of 4 d culture was in-
on Carotenoids, Kyoto, p. 41, 1990) as a lipophilic oxygen oculated into 100 ml of fresh medium in a 200 ml Erlen-
quencher. meyer flask. The flask was incubated at 20°C under 12 h-
Although astaxanthin is an abundant carotenoid in ma- light and 12 h-dark illumination condition (1.5 klx, fluores-
rine animals, especially crustacea, only a few microorgan- cent light, LH-200-RD or LH-500-RD, Nippon Medical
isms have been reported to synthesize astaxanthin (3). and Chemical Instruments, Co. Ltd., Tokyo). The flask
Phaffia rhodozyma, which is one of the possible candi- was shaken manually once a day. The modified medium
dates for industrial scale production of astaxanthin, has (pH 7.0) consisted of sodium acetate, 14.6 mM; L-aspar-
been extensively investigated for its carotenoid production agine, 2.7 mM; yeast extract, 0.5 g/l; tris-(hydroxymethyl)-
(4, 5), and for the feasibility of the astaxanthin as a feed aminomethane, 6.2 mM; MgC12" 6H2O, 0.640 mM; FeSO4.
additive for salmonid aquaculture (1). Few research, 7H20, 0.216mM and MnC12-4H20, 0.808mM using
however, has been published on practical carotenoid deionized water. The same cultivation conditions as used
production by a green unialga Haematococcuspluvialis (6) for the basal medium were used for the modified medium
due to several disadvantageous characteristics of the alga: except that the illumination condition was changed to
its slow growth, low growth temperature, and light require- 4.5 klx for 24 h continuous lighting.
ment for cultivation. Recently, commercial productions of Analyses The carotenoids and chlorophylls were ex-
E-carotene and glycerol by a green microalga Dunaliella sa- tracted with 90%0 acetone for 1 h after grinding the algal
lina have been successfully employed in open ponds as well cells with a pestle. The total carotenoid concentrations
as biomass production of Spirulina in several countries (7, were determined at A480 using the absorption coefficient
8). In a reproducible laboratory experiment, we first stud- A ~ - - 2 , 5 0 0 (10). The total chlorophyll concentration was
ied the algal medium composition for improved algal cell calibrated as chlorophyll-a for the major chlorophyll com-
growth and higher carotenoid production. Then, the two ponent according to Strickland and Parsons (10). For fur-
media developed in this study were characterized in terms ther analysis of carotenoids, the cells were treated with
of the algal growth, astaxanthin biosynthesis, and the algal 40% acetone overnight, and the residue was extracted with
morphological changes. In addition, supplementation of 70°//ooacetone. Solvent was then removed by vacuum evapo-
ferrous ion and acetate into the basal medium under en- ration with the addition of benzene under nitrogen gas.
hanced light intensity during the culture was shown to be The carotenoids were applied on silica gel plate (Kieselgel
stimulative both for algal cell growth and carotenoid for- 60, 0.2 mm thickness, Merck), and developed in the sol-
mation. vent system (acetone: n-hexane, 3 : 7 v/v) in the dark to
prevent photodegradation of the carotenoids. The carote-
MATERIALS AND METHODS
noids were identified with the reported Rf value (11) and
the authentic carotenoids. The ratio of the carotenoids was
Algal strain H. pluvialis Flotow NIES-144 obtained determined by absorption at 545 nm with a TLC scanner
from the National Institute for Environmental Studies (CS-930, Shimadzu, Kyoto). Thus, the concentration of
(NIES), Japan was used in this study. each astaxanthin form was calculated based on the propor-
tion of the carotenoid composition. The algal cell growth
* Corresponding author. was determined either by cell number counting with hae-
335
336 KOBAYASHIET AL. J. FERMENT.BIOENG.,

macytometer, or by dry cell weight (DCW) after being TABLE 1. Fractionof the carotenoids produced in the basal
filtrated on cellulose nitrate (0.45 pm, Toyo Roshi), and and the modified media by 1t. pluvialis
drying at 80°C for 3 h. Acetate was measured by gas chro- Media
matography (GC-8, Shimadzu) equipped with a 3 m long Carotenoid (%)
glass column packed with polyethyleneglycol 6000. Both Basala Modified
~ Reported (11)
the injector and the detector were kept at 200°C, and the Astaxanthin
column oven was isothermally operated at 170°C. The sam- Monoester 74 69 76
ple from the medium was acidified with 6 N HC1 to convert Diester 10 20 7
acetate ion to the acid form prior to the analysis. Free form n.d) n.d. 1
Total 84 89 84
Lutein 9 1 7
RESULTS AND DISCUSSION Adonirubin ester 6 9 3
Violaxanthin md. n.d. 2
Growth and astaxanthin production Since H. pluvia- Neoxanthin n.d. n.d. 1
lis showed slow autotrophic growth on C medium recom- fl-Caroten¢ n.d. n.d. 1
mended by the NIES, several media were examined for al- a The cultivation conditions were described in Fig. 1.
gal growth and astaxanthin production. In Ondratscheck b n.d.: Not detectable.
medium (12), the alga changed the cellular color from
green to red without a significant increase in cell number,
suggesting the chlorophyll retardation and carotenoid for- as that in the basal medium. Secondly, while the carote-
mation in the cell. In Pringsheim medium (13) as well, the noid biosynthesis was actively operated, the chlorophyll
alga showed poor growth. However, the alga grew well in synthesis was apparently inhibited in the modified medi-
terms of cell number and cell mass per liter in the basal um, resulting in the unvarying appearance of the red
medium which was used in Euglena cultivation (9). Thus, colored cells. Thirdly, compared to the basal medium, H.
this basal medium was employed as the vegetative growth pluvialis cells did not increase in cell number in the
medium in the present study. Typical time courses of the modified medium. Instead, approximately 10°A of the
cell growth and the carotenoid formation are shown in population of algal cells grew in size up to 50-70 pm in
Fig. la. The green cells exhibited good growth in responce average diameter which was two to three times larger than
to acetate consumption. Acetate was heterotrophically that of the basal medium fFig. 2c). The rest of the popula-
assimilated only under illumination. When acetate was tion completely turned into colorless ceils (Fig. 2d) indicat-
replaced with other carbon sources such as lactate, pro- ing drastic physiological changes. This bleaching effect of
pionate, butyrate, citrate, succinate, oxalate, malate, glu- the modified medium remains to be elucidated. Fourthly,
cose, fructose, xylose, galactose, mannose, sucrose, man- in the basal medium, the alga carried out the typical vegeta-
nitol, and glycerol, the alga showed insignificant growth tive growth of other green algae indicating several morpho-
under illumination. logical changes in the life cycle (Fig. 2a,b) in which vegeta-
In order to increase astaxanthin content, the basal medi- tive oval cells were actively swimming with biflagella, and
um was modified to a metal-rich medium, termed as the round zygote called akinetes or cysts were formed either by
modified medium, because preliminary experiments in our h0mothallic or by heterothallic mating. On the contrary,
laboratory showed that iron and manganese ions increased in the modified medium, H. pluvialis cells seemed to enter
the carotenoid concentration. The alga grown in the the cyst stage within a shorter period (5-6 d) compared
modified medium exhibited several unique characteristics with more than 20 d required in the basal medium, and in
(Fig. lb). Firstly, the carotenoid synthesis rate was much fact, the algal cyst.s contained a high level of carotenoids.
enhanced in the modified medium. The final carotenoid From these observations, it could be suggested that the
content was 20 mg per g dry cell, which was twice as much alga was obliged to proceed along the irregular cell cycle in
the modified medium. Furthermore, it was very likely that
these morphological shifts could be attributed to the high
10 concentrations of metals such as iron and manganese in
~ 8
the modified medium. In addition, only slight differences
I I I ,~ were detected in the chemical forms of astaxanthin pro-
2O I t • ;t duced in the two media (Table 1). There was negligible
E difference from the reported values as well, although H.
o= ~ 15 15~
10~-~
o E
TABLE 2. Stimulation of carotenogenesis by metabolites involved
5 in mevalonate biosynthetic pathway
| i •

8
Carotenoid Chlorophyll DCWb
Additiona
~ 6~4 lo~N
None (control)
(mM) (rag//) (mg/g cell) (rag//)
14.8 19.7 3.5
(g//)
0.75
2 Mevalonate
Isopentylalcohol 3
3 18.2
15.3
20.7
18.4
5.6
4.4
0.88
0.83
0 2 4 6 Pyruvate 12 20.6 26.8 0.8 0.77
Culture time (d) Malonate 3 17.7 23.0 2.3 0.77
L-Leucine 3 11.2 19.6 2.4 0.57
FIG. 1. Growth and carotenoid formation of Haematococcus Dimethylacrylate 6 16.9 24.1 3.0 0.70
pluvialis in the basal medium (a) and the modifiedmedium (b). Sym-
bols: n, pH; o, chlorophylls; O, carotenoids; O, acetate; A, cell a The alga was cultivated for 8 d in the modified medium contain-
number. Illumination conditions: (a) 12 h-light/12 h-dark at 1.5 klx; ing the each metabolite.
(b) 24 h continuous lighting at 4.5 klx. b DCW: Dry cell weight.
VoL. 71, 1991 ASTAXANTHIN PRODUCTION BY A GREEN ALGA 337

ol

FIG. 2. Light microphotographs of H. pluvialis in the basal medium (a, b) and the modified medium (c, d). (a) Green oval cells with
biflagella at 4 d; (b) round cells with a red eyespot at 30 d; (c) enlarging round cells at 8 d; (d) bleached cells at 8 d. The bar indicates 20/zm.
Illumination conditions: (a) (b) 12 h-light/12 h-dark at 1.5 klx; (c) (d) 24 h continuous lighting at 4.5 klx.

FIG. 5. Morphological changes of H. pluvialis in the supplementation of ferrous ion and acetate into the basal medium. Ferrous ion and
acetate were added to 450/AM and 45 raM respectively into the basal medium at 4 d of cultivation. Illumination was shifted as described in Fig. 4.
(a) At the begining after the addition; (b) at 2 d later; (c) at 4 d later; (d) at 6 d later. The bar indicates 20/~m.
338 KOBAYASHIET AL. J. FERMENT.BIOENO.,

~. 25 modified medium. Furthermore, when acetate and pyru-

r IC' i l°- vate were employed together, these two substrates were
20 i~ O, 8 ~ stimulative on both the cell growth and the carotenoid
production (Fig. 3c).
15 ( 0,6~ Although Bejarano et al. showed that the synthesis of/3-
carotene in Phycomyces blakesleanus was stimulated by/3-
I0 O,4 "~ ionone and its analogues such as dimethyl phthalate (16),
0,2 ~ /3-ionone and its analogues inhibited completely carote-
noid synthesis in H. pluvialis (data not shown).
o( , 0 "" Two-stage batch culture A dense algal cell culture
20 40 60 0 I0 20 30 40 0 3 6 9 12 would be required to enhance the astaxanthin productiv-
Acetote (raM) PYruvete (raM) PYruvQte (raM) ity. The two media designed in the present study, however,
FIG. 3. Effect of acetate and pyruvate concentrations on seem unsuitable for this purpose because the basal medium
carotenogenesis by H. pluvialis in the modified medium. Substrates was appropriate for the vegetative growth, and the modi-
employed were (a) acetate, (b) pyruvate and (c) acetate 15 mM plus fied medium was for the carotenoid synthesis. Thus, we
pyruvate. The culture period was 8 d. Symbols: A, cell; O, caro- attempted to carry out a two-stage batch cultivation: for
tenoids; U, chlorophylls. Illumination condition: 24 h continuous the first-stage, to attain a high cell concentration in the
lighting at 4.5 klx.
basal medium (Fig. la), and for the second-stage, to en-
hance carotenoid formation by the injection of ferrous ion
pluvialis was cultivated autotrophically in 10% Z8 medi- with acetate into the middle of the first-stage culture. In
um (11). this two-stage culture, since algal cells might obtain less
Effect of several precursors To enhance the produc- light intensity per cell in the second-stage, the illumination
tivity of the astaxanthin formation in the alga, several pos- was switched from 12 h-light/12h-dark at 1.5 klx in the
sible precursors for carotenoid synthesis were added into first-stage to 24 h continuous lighting at 8.6 klx in the
the modified medium to examine the stimulation effect. second-stage.
Mevalonate, which is a key intermediate in forming 3- In the supplementation of ferrous ion, acetate was also
hydroxy-3-methylglutaryl-CoA (HMG-CoA) in isoprenoid supplied to avoid its depletion. The algal cell growth and
compound biosynthesis, like steroids and carotenoids, was the carotenoid synthesis were dramatically enhanced with
effective in carotenoid formation as shown in Table 2. higher concentrations of ferrous ion up to 450/~M (Fig.
Moreover, pyruvate, malonate, and dimethylacrylate 4a, c). Furthermore, while assimilation of acetate was ac-
seemed to promote carotenoid synthesis. Although L-leu- celerated, the chlorophyll content was decreased (Fig. 4b,
cine is a precursor of HMG-CoA (14), it seemed to inhibit d). In this supplementation experiment, H. pluvialis grew
cell growth. Several intermediates in the TCA cycle such as into either enlarged cysts or bleached cells as shown in Fig.
succinate, fumarate, malate, and oxaloacetate all had an in- 5, which was similar to the morphological changes in the
hibitory effect at a level of 10 mM in the modified medium, modified medium (Fig. 2c, d). Thus, we were able to attain
while the /3-carotene synthesis in Blakeslea trispora was higher astaxanthin production by the addition of ferrous
facilitated by these TCA intermediates (15). Since pyruvate ion with acetate in the two-stage batch culture. It has been
seemed to be a good alternative for acetate, the effect of reported that unfavorable environmental conditions such
pyruvate concentration on the carotenoid synthesis was in- as nitrogen deficiency are required for secondary carote-
vestigated in the modified medium (Fig. 3a, b). Although noid biosynthesis (6). Astaxanthin biosynthesis in H. plu-
acetate was inhibitory at high concentrations of more than vialis, however, might be triggered by ferrous ion accompa-
30 mM, pyruvate has turned out a better substrate because nied with the morphological changes mentioned above
pyruvate was not only more stimulative on the cell growth (Fig. 5). The physiological role of ferrous ion observed in
and the carotenoid synthesis than acetate was, but also less the supplementation experiment is under investigation.
inhibitory as a substrate at high concentrations in the

30 -I 2 , 0
a) ' b) "' c)

50 1.5

qO 1,0 "~
~ lO
30 0,5

L 2O 0
0 2 4 6 0 2 4 6 0 2 4 6 0 2 4 b

C u l t u r e tLme (d)
FIG. 4. Supplementationof ferrous ion and acetate into the basal medium under the enhanced illumination. Ferrous ion and acetate were
added to the basal medium at 4 d of the cultivationscaled as zero time. Concentrationsemployedwere0 ( • ), 150 ( o ), 300 (zx),450 (<>)and 600 (v)
for ferrous ion (uM) and 45 mM for acetate. At the supplementation, the illumination conditions were shifted from 12 h-light/12 h-dark
(1.5 klx) to 24 h continuous lighting (8.6 klx). (a) Carotenoids; (b) chlorophylls; (c) dry cell weight; (d) acetate.
VoL 71, 1991 ASTAXANTHIN PRODUCTION BY A GREEN ALGA 339

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