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JOURNALOF FERMENTATION

ANDBIOENGINEERING
Vol. 84, No. 1, 94-97. 1997

Morphological Changes in the Life Cycle of the Green


Alga Haematococcus pluvialis
MAKIO KOBAYASHI,*
YOSHIRO KURIMURA,
TOSHIHIDE
NAOMICHI NISH10,2 AND YASUNOBU TSUJI

KAKIZON0,2

Research Laboratory of Higashimaru Shoyu Co. Ltd., 100-3 Tominaga, Tatsuno, Hyogo 679-41 and Department of
Fermentation Technology, Faculty of Engineering, Hiroshima University, Higashi-Hiroshima 724,2 Japan
Received 10 January 1997IAccepted 28 April 1997

A 2-week model life cycle of the green alga Haematococcuspluvialis was constructed, consisting of four cell
stages: vegetative cell growth, encystment, maturation, and germination. Each algal cell stage could be distinguished by the ratio of pigments (carotenoid/chlorophyll) and the intracellular protein content. Using the
culture system developed, light was shown to be essential for both carotenogenesis and cell differentiation
(encystment and germination). The results also suggested that H. pluvialis has a novel photosynthesis-dependent system of carotenogenesis regulation.
[Key words:

astaxanthin,

encystment,

germination,

Haematococcuspluvialis, life cycle, maturation]


the acetate-supplemented
culture at a final concentration
of 450,uM to prepare mature (carotenoid-rich)
cyst cells
(15). The supplementation
cultures were incubated
at
20C under continuous illumination (125 pmol quanta.m-2+
s-l) as described previously (15).
After another 3 d, mature cyst cells in the Fe2+-supplemented
culture were harvested by centrifugation
at
2,OOOxg for lOmin, and washed twice in fresh basal
medium. The washed mature cysts were resuspended in
100 ml fresh basal medium. Then, a lo-ml portion of the
mature cysts was transferred to 1OOml fresh basal medium in a flask. The flask was incubated at 20C under a
12-h light/lZh
dark illumination
cycle (25 ,umol quanta.
rnp2. s- ) as the basal culture of vegetative cells.
To investigate the regulation of the life cycle of H.
pluvialis, the following inhibitors obtained from Sigma
Chemical Co. (St. Louis, MO, USA) were used as described previously
(13, 15): a transcriptional
inhibitor
(actinomycin
D) at a final concentration
of 10 ,ug.mlVl;
two translational
inhibitors (cycloheximide for cytoplasmic protein synthesis and chloramphenicol
for organellic
protein synthesis, at 0.3 /-lg.ml- and 50 pg+rnl-, respectively); 3-(3,Cdichlorophenyl)-1,1-dimethylurea
(DCMU),
a specific inhibitor
of electron flow from photosystem
(PS) II to the plastoquinone
pool (16), at 20 PM; and 2,5dibromo-3-methyl-6-isopropyl-p-benzoquinone
(DBMIB),
a specific inhibitor of electron flow from plastoquinone
to cytochrome (Cyt) f (16), also at 20 ,QM.
The algal cell number was counted with a hemacytometer. For the protein assay, algal cells were suspended in
2 M NaOH for 1 h on ice as described by Whitelam and
Codd (17) and the alkaline-solubilized
protein was determined by the Bradford method (18) with bovine serum
albumin
as the standard.
The intracellular
pigments
carotenoid
and chlorophyll
were extracted with 90%
(v/v) acetone, and assayed as described previously (5,
15). All analyses were performed in triplicate cultures,
and the means are shown.
Morphological changes of H. pluvialis
The model
life cycle of H. pluvialis using acetate media was divided
into four cell stages, as illustrated in Fig. 1: I, vegetative
cell growth; II, encystment (vegetative to immature cyst
cells); III, maturation
(immature to mature cyst cells);

Astaxanthin
(3,3-dihydroxy-P,
p-carotene-4,4-dione),
a red ketocarotenoid,
is not only used as a pigmentation
source in marine fish aquaculture (l), but also has potential clinical applications
due to its higher antioxidant
activity than p-carotene and vitamin E (2, 3). The green
unicellular
alga Haematococcus pluvialis is a potent
producer of astaxanthin
(4, 5). A number of recent
studies have suggested that astaxanthin
biosynthesis
in
H. pluvialis is associated with the formation
of cyst
cells (encystment) in the resting stage under autotrophic (4,
6-8) and mixotrophic conditions (5, 9-11). The morphological change from vegetative to enlarged thick-walled cyst
cells has been reported to take several weeks under autotrophic conditions
(4, 7), and in nitrogen-deficient
(10)
and phosphate-deficient
(11) acetate media. However, we
previously showed (12) that a high acetate content in the
medium induced encystment
within several days under
mixotrophic
conditions.
We have also examined various
environmental
factors influencing growth and carotenoid
biosynthesis
in H. pluvialis, including media composition (5), growth kinetics (13), light conditions (14), and
environmental
stress (15). In the present study, we constructed a 2-week model life cycle of H. pluvialis and investigated the mechanisms of the morphological
changes
that occurred.
The freshwater alga H. pluvialis Flotow NIES-144 was
obtained from the National Institute for Environmental
Studies, Tsukuba,
Japan. An acetate (15 mM) basal
medium was used as described previously (5). For the
basal culture, 10ml of a 4-d-old culture was inoculated
into 100 ml fresh basal medium in a 200-ml Erlenmeyer
flask. The flask was incubated at 20C under a 12-h light/
12-h dark illumination
cycle at 25 pmol quanta-mp2.
s-l
(white fluorescent lamp) as described previously (5).
The 4-d culture (vegetative cells in the exponential
growth phase) was used for the subsequent
two-stage
supplementation
culture. Sodium acetate solution (2.25
M, pH 7.0) was first added to the 4-d basal culture at a
final concentration
of 45 mM to prepare immature (carotenoid-poor) cyst cells (12). Then, after a further 2 d, ferrous sulfate solution (22.5 mM, pH 1.5) was added to

* Corresponding author.
94

NOTES

VOL. 84, 1997

Vegetative Cell

stage

111

II

IV-

95
0
P

Ireah medium

!!
=

7
6

2
g

:!/
52
1
0

Immatu%
FIG. 1.

Cyst

Schematic diagram of the model life cycle of H. pluviulis.

TABLE 1.
Cell stage&

Protein
@g/cell)
Lightc
None
+Actinomycin D
+ Chloramphenicol
+ Cycloheximide
+DCMU
+ DBMIB
Dark

None

60

200
250
150

40
50
30

iiIi
5

100

20

50

10
0

1011121314

E
8
if
f
g

Culture time(d)
FIG. 2. Changes in the contents of protein ( q ), carotenoid (a),
and chlorophyll (A), cell number (A) and the ratio of carotenoid/
chlorophyll (0) in the life cycle of H. pluviulb. (A) Cell number and
pigment ratio; (B) intracellular contents. Each cell stage (I-IV) in
the life cycle of H. pluviulis is shown in Fig. 1. Culture times: O-4 d,
vegetative cell growth (I); 4-6 d, encystment (II); 6-9 d, maturation
(III); 9-14d, germination and vegetative cell growth (IV-rI). The
three arrows indicate the times of acetate addition (45 mM), Fe2+
addition (450,uM), and transfer to fresh medium, respectively.
Light conditions: O-4 d, 12-h light/l2 h-dark illumination cycle (25
pmol quanta-m-*.sst);
4-9 d, continuous illumination (125 pmol
quanta.m-2.s~1); 9-14 d, 12-h light/l2 h-dark illumination cycle (25
,nmol quanta. m-2. SC).The means of three replicate experiments I
standard deviation as shown.

maturation,
and germination
are usually assessed by
microscopic
observation.
In this study, we determined
each algal cell stage using the intracellular
protein and
pigments contents. Since carotenoid accumulated in cyst
cells only on maturation,
the ratio of intracellular
carotenoid/chlorophyll
was considered a good parameter for
distinguishing
among vegetative (green), immature cyst
(brown), and mature cyst (red) cells. The pigments ratios
were about 0.5, 1.0, and 7.0 in vegetative, immature, and

Effects of light and inhibitors on each cell stage in the life cycle of H. pluvialis

I (0-4d)
at 4d

Culture timea

300

IV, germination
(mature cyst to vegetative cells). The
changes in the intracellular
contents and cell number in
the algal life cycle are shown in Fig. 2. Ellipsoidal vegetative cells were capable of actively swimming with two
flagella and of increasing in number (O-4 d culture). Supplementing the vegetative cell culture with a high level of
acetate at 4 d induced the algal cells to become spherical
immotile cyst cells (4-6 d culture). During maturation
(6-9 d culture), carotenoid biosynthesis in cyst cells was
significantly enhanced by the addition of FeZ+ at 6 d. When
mature cysts were transferred
to fresh medium at 9 d,
intracellular daughter cells were released from the mature
cyst cells into the medium, and vegetative cells regenerated from daughter
cells grew mixotrophically
(9-14 d
culture). In this way, the life cycle of H. pluvialis was
completed within only 2 weeks in our experimental culture
system.
During the life cycle of the alga, vegetative cells contained high levels of chlorophyll
and protein but had
very low carotenoid
contents, whereas encystment
was
accompanied by the degradation of chlorophyll and protein. The maturation
of cyst cells was accompanied
by
enhanced carotenoid biosynthesis and accelerated protein
degradation.
Germination
coincided with chlorophyll and
protein syntheses, and carotenoid degradation. Encystment,

o!
e
a

II (4-6 d)

III (6-9 d)

at 6d

at 9d

Car/Chlb

Protein
(pglcell)

270*23
NDd
250&25
NDd
254124
260 + 30

0.4kO.l
ND*
0.5t0.1
NDd
0.3kO.l
0.4fO.l

250220

0.520.1

IV-*1 (9-14 d)
at 14d

Car/ChP

Protein
(pg/cell)

Car/Chlb

13*5
25t9
29f5
33+8
145+-20
20*5

7.OkO.7
6.OkO.5
7.OkO.6
6.5kO.5
0.9t0.3
7.520.8

281+25
15c5
20F4
13+5
45+10
40t9

0.3i-0.1
5.520.5
5.9kO.6
5.7kO.5
5.510.5
5.2kO.4

140+ 15

0.7kO.3

5OilO

5.310.5

Car/ChP

Protein
(pg/cell)

150+15
251+30
145+20
244+25
255?28
240? 19

l.lkO.2
0.3LO.l
l.lkO.2
0.4kO.l
0.4t0.1
0.4fO.l

248&30

0.5kO.l

a Each cell stage and indicated times are as shown in Fig. 2.


b Ratio of carotenoid/chlorophyll.
c Cell treatments: light conditions were as shown in Fig. 2. In each cell stage, cells were cultured for the indicated times in the presence of
inhibitors. Inhibitor concentrations: actinomycin D, 10 pg.ml-t; chloramphenicol, 50 ,ug.ml-; cycloheximide, 0.3 /-g.ml- r; DCMU, 20 pM;
DBMIB, 20 /IM.
d Not determined because of no growth.

96

J. FERMENT.BIOENG.,

KOBAYASHI ET AL.

mature cyst cells, respectively.


Effects of light and inhibitors on the algal life cycle
We have already shown that vegetative cells of H. pluviuZis can grow heterotrophically
on acetate in the dark
(13). We investigated
the light requirement
and regulation of each cell stage during the model life cycle of H.
pluvialis using various inhibitors.
The results are shown
in Table 1. Green vegetative cells grew heterotrophically
on acetate in the dark (about 3 x lo5 cells/ml), as well as
mixotrophically
on acetate in the light (about 5.5 x 10
cells/ml).
Although
photosynthesis
was completely inhibited by either DCMU or DBMIB, algal vegetative cell
growth was retained at the heterotrophic
level in the
light. Actinomycin
D and cycloheximide
completely inhibited vegetative cell growth in the light, whereas vegetative cells grew at the heterotrophic
level in the presence
of chloramphenicol.
Neither cell differentiation
(encystment
and germination) nor maturation
(carotenogenesis)
occurred in the
dark. With addition of acetate to H. pluvialis in the
vegetative growth phase, cysts were formed within 48 h
in the light. This encystment was blocked by actinomytin D or cycloheximide,
but was unaffected by chloramphenicol, suggesting that encystment is regulated at the
transcriptional
level of algal chromosomal
genes, and
probably does not involve organellic genes. No encystment was observed in the presence of either DCMU or
DBMIB, indicating that the induction of encystment by
a high C/N ratio might require photosynthesis.
Carotenogenesis
in cyst cells was enhanced by FeZ+
both in the presence and absence of transcriptional
or
translational
inhibitors
in the light. To further investigate photo-dependent
carotenogenesis
in the cyst cells,
two electron flow inhibitors of photosynthesis
were used
(16). Fe2+-enhanced
carotenogenesis
in cyst cells was inhibited by DCMU but not by DBMIB, suggesting that
plastoquinone
might be involved in carotenoid biosynthesis of H. pluvialis. Recently, it was reported that plastoquinones can be substituted
for molecular oxygen as a
terminal electron acceptor in the carotenoid biosynthetic
pathway in phytoene desaturation
in chromoplasts of the
daffodil, Narcissus pseudonarcissus (19, 20). Moreover,
Cyt f has been shown to be selectively lost in red cyst
cells of H. pluvialis (ll), while selective destabilization
of Cyt f occurred during gamete induction in Chlamydomonas (21). Therefore, in cyst cells of H. pluvialis, instead of impairment
of the linear electron flow between
PS II and PS I, the plastoquinone
pool may function as
an electron crossover point between photosynthesis
and
carotenoid biosynthesis.
When mature cyst cells prepared within only 5 d (induction for 2 d followed by activation
for 3 d) were
transferred to a fresh basal medium, intracellular
daughter cells were rapidly released from the mother cells (cyst
cells) into the medium in the light. We observed the
same results regarding germination
using 2-month-old
cyst cells as described by Lee and Ding (22). Vegetative
cells regenerated from daughter cells grew mixotrophically or heterotrophically
on acetate in the light or dark
(data not shown). Germination
was inhibited
by the
three antibiotics
tested, actinomycin
D, cycloheximide,
and chloramphenicol.
These results suggest that, unlike
the case with cyst formation, cell differentiation
from the
resting to vegetative growth stage is regulated at the
transcriptional
level of both chromosomal
and organellic
genes. Germination
as well as encystment was inhibited

by the two photosynthesis


inhibitors.
From these results, it is suggested that light is essential
for the life cycle of H. pluvialis, particularly
for cell
differentiation
(encystment and germination)
and maturation (carotenogenesis).
The fact that these metabolic
events were all inhibited by DCMU suggests that photosynthesis is an absolute requirement
for their occurrence. Since light-dependent
carotenoid
formation
has
been reported in many microorganisms
(23), such photosynthesis-dependent
induction
and activation
indicate
that H. pluvialis has a novel photo-dependent
system for
the regulation of carotenogenesis.

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NOTES

97

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