Professional Documents
Culture Documents
ANDBIOENGINEERING
Vol. 84, No. 1, 94-97. 1997
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,
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
Vegetative Cell
stage
111
II
IV-
95
0
P
Ireah medium
!!
=
7
6
2
g
:!/
52
1
0
Immatu%
FIG. 1.
Cyst
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
96
J. FERMENT.BIOENG.,
KOBAYASHI ET AL.
REFERENCES
1. Renemann, J. R.: Microalgae aquaculture feeds. J. Appl.
Phycol., 4, 233-245 (1992).
2. Mikl, W.: Biological functions and activities of animal carotenoids. Pure Appl. Chem., 63, 141-146 (1991).
3. Palozze, P. and K&sky, N. I.: Astaxanthin and canthaxanthin
are potent antioxidants in a membrane model. Arch. Biochem.
Biophys., 297, 291-295 (1992).
4. Borowitzka, M. A., Huismao, J. M., and Osborn, A.: Culture
of the astaxanthin-producing green alga Haematococcus pluvialis. I. Effects of nutrients on growth and cell type. J. Appl.
Phycol., 3, 295-304 (1991).
5. Kobayashi, M., Kakiiono, T., and Nagai, S.: Astaxanthin
production by a green alga, Haematococcus pluvialis accompanied with morphological changes in acetate media. J. Ferment.
Bioeng., 71, 335-339 (1991).
6. Boussiba, S. and Vonshak, A.: Astaxanthin accumulation in
the green alga Haematococcus pluvialis. Plant Cell Physiol.,
32, 1077-1082 (1991).
7. Harker, M., Tsavalos, A. J., and Young, A. J.: Autotrophic
growth and carotenoid production of Haematococcus pluvialis
in a 30 liter air-lift photobioreactor.
J. Ferment. Bioeng., 82,
113-118 (1996).
8. Lee, Y. K. and Sob, C. W.: Accumulation of astaxanthin in
Haematococcus lacustris (Chlorophyta). J. Phycol., 27, 575577 (1991).
9. Droop, M. R.: Carotenogenesis in Haematococcus pluvialis.
Nature, 175, 42 (1955).
10. Zlotnik (Shmerler), I., Sokenik, A., and Dubinsky, Z.: Physiological and photosynthetic changes during the formation of red
aplanospores in the chlorophyte Haematococcus pluviatis. J.
Phycol., 29, 463-469 (1993).
11. Tan, S., Cunningham, F. X., Youmans, M., Grabowski, B.,
Sun, Z., and Gantt, E.: Cytochrome f loss in astaxanthinaccumulating red cells of Haematococcus pluvialis (Chlorophyceae): comparison of photosynthetic activity, photosynthetic enzymes, and thylakoid membrane polypeptides in red
and green cells. J. Phycol., 31, 897-905 (1995).
12. Kakizono, T., Kobayasbi, M., and Nagai, S.: Effect of carbon/nitrogen ratio on encystment accompanied with astaxanthin formation in a green alga, Haematococcus pluviaZis. J.
Ferment. Bioeng., 74, 403-405 (1992).
13. Kobayashi, M., Kakiiono, T., Yamagucbi, K., Nisbio, N., and
Nagai, S.: Growth and astaxanthin formation of Haematococcus pluvialk in heterotrophic and mixotrophic conditions. J.
Ferment. Bioeng., 74, 17-20 (1992).
14. Kobayasbi, M., Kakizono, T., Nishio, N., and Nagai, S.:
Effects of light intensity, light quality, and illumination cycle
on astaxanthin formation in a green alga, Haematococcus
pluvialis. J. Ferment. Bioeng., 74, 61-63 (1992).
15. Kobayashi, M., Kakizono, T., and Nagai, S.: Enhanced carotenoid biosynthesis by oxidative stress in acetate-induced cyst
cells of a green unicellular alga, Haematococcus pluvialis.
Auul. Environ. Microbial., 59, 867-873 (1993).
16. Escoubas, J-M., Lomas, -M.; LaRoche, J.; and Falkowski,
P. G.: Light intensity regulation of cab gene transcription is
signaled by the redox state of the plastoquinone pool. Proc.
NOTES
97
linked to a respiratory redox pathway in Narcissus pseudonarcissus chromoplast membranes. Involvement of a 23-kDa oxygen-evolving-complex-like protein. Eur. J. Biochem., 233, 864-872
(1995).
21. B&e, L. and Wollman, F-A.: Evidence for a selective destabilization of an integral membrane protein, the cytochrome b6/f
complex, during gametogenesis in Chlumydomonas reinhordtii.
Eur. J. Biochem., 204, 327-336 (1992).
22. Lee, Y. K. and Ding, S. Y.: Cell cycle and accumulation of
astaxanthin in Haematococcus
Iucustris (Chlorophyta).
J.
Phycol., 30, 445-449 (1994).
23. Rau, W.: Mechanism of photoregulation of carotenoid biosynthesis in plants. Pure Appl. Chem., 57, 777-784 (1985).