Biosci. Biotechnol. Biochem.
, 68 (7), 15941597, 2004
Note
Growth and Carotenoid Production of Thraustochytrium sp. CHN-1
Cultured under Superbright Red and Blue Light-emitting Diodes
Yukiho Y AMAOKA,1; y Marvelisa L. C ARMONA,2 and Shinji O OTA3
1
National Institute of Advanced Industrial Science and Technology,
2-2-2 Hirosuehiro, Kure, Hiroshima 737-0197, Japan
2
School of Biosphere Sciences, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima 737-8528, Japan
3
Instrument Center for Chemical Analysis, Hiroshima University,
1-4-4 Kagamiyama, Higashi-Hiroshima 737-8528, Japan
Received February 16, 2004; Accepted April 15, 2004
Isomers of astaxanthin produced by Thraustochytri-
um sp. CHN-1 are identied as (3S,3S0 )-trans-astaxan-
thin, (3R,3R0 )-trans-astaxanthin and (3S,3S0 )-cis-astax-
anthin by chirality column HPLC, and 1 H and 13 C
NMR. We studied the eects of light generated by
superbright blue, red and near-red LEDs on the growth
and carotenoid production of Thraustochytrium sp.
CHN-1. Thraustochytrium sp. CHN-1 responded to blue
LEDs light: It produced carotenoid pigments (astaxan-
thin)
Key words: Thraustochytrium; light-emitting diode;
(3S,3S0 ) trans-astaxanthin
The genus Thraustochytrium contains unicellular,
zoospore-producing species of historically uncertain
taxonomic anity that have been isolated mainly from
coastal marine environments in sub-tropical and tropical
zones.1) Thraustochytrium species have attracted interest
recently because they produce docosahexaenoic acid
(22:6, DHA), eicosapentaenoic acid (20:5, EPA), and
docosapentaenoic acid (22:5, DPA).2) Thraustochytrium
sp. CHN-1, a DHA and carotenoid (astaxanthin, pheno-
coxanthin, canthaxanthin, echinenone, and
-caro-
tene3))-producing strain, was isolated from sea water
samples collected from the coastal area of Nagahama
(Hiroshima Prefecture.) in the Seto Inland Sea of Japan.
In the yeast Phaa rhodozyma,4) Agrobacterium aur-
auticum5) and algal Haematococcus pluvialis,6) astax-
anthin is the major carotenoid. For utilization of
Thraustochytrium sp. CHN-1 as a feed for nutritional
enrichment of rotifer and Artemia, a large volume
culture of this strains cells was evaluated in sea water.
Many studies suggest that astaxanthin has about 10-fold
higher antioxidant activity than other carotenoids.7)
Astaxanthin exists mainly as free (3S,3S0 )-trans-astax-
anthin in Thraustochytrium sp. CHN-1.3) But astaxan-
thin in Thraustochytrium sp. CHN-1 was not judged
y
To whom correspondence should be addressed. Tel: +81-823-72-1934; Fax: +81-823-73-3284; E-mail: yamaoka-yu@aist.go.jp
using 1 H and 13 C NMR. This communication describes
the identication of carotenoids by spectral means
(chirality column HPLC and 1 H and 13 C NMR) together
with authentic compounds. No information exists in the
literature regarding light sources for growth of cultured
Thraustochytrium. Light emitting diodes (LEDs) possess
features that are lacking in frequently used radiation
sources such as uorescent lighting, metal halide. The
most attractive features of LEDs are their small mass,
volume, solid state construction, and long life. They
have been used recently in several plant cultures because
of their wavelength specicity and narrow bandwidth.8)
This report describes the growth and carotenoid pro-
duction of Thraustochytrium sp. CHN-1 cultured under
superbright red and blue LEDs.
Pure slant cultures of Thraustochytrium sp. CHN-1
were grown for 48 h on GPY media containing 20 g
glucose, 10 g peptone, 5 g yeast extract, and 20 g agar
per liter of half salt concentration seawater. Vegetative
cells were inoculated into 100-ml Erlenmeyer asks, one
loopful each. The asks contained 30 ml of the follow-
ing sea medium: 3% (wt/v) glucose, 0.1% (wt/v) yeast
extract, and 0.1% (wt/v) polypeptone.
The liquid culture was grown under 1500 lux intensity
uorescent light, and light from red LEDs (max 660
nm), blue LEDs (max 470 nm) and near-infrared LEDs
(max 730 nm). They were rotary shaken at 120 rpm at
23
C. At various times, from 415 d of culture, biomass
and carotenoid contents were determined for three
randomly selected asks, one from each group. Cells
in the liquid cultures were harvested by centrifugation at
2; 000  g for 20 min, and the supernatants were then
discarded. Cell pellets were rinsed with 10 ml of cold
reconstituted seawater twice and then freeze-dried using
a Yamato NEOCOOL type freeze-dryer, then stored at
20
C prior to extraction. Cell dry weight concentra-
tions were determined according to the gravity weight
procedure. All experiment were done in triplicate, and
 Production of Carotenoids by Thraustochytrium sp. 1595

Fig. 1. HPLC Chromatogram of Thraustochytrium sp. CHN-1 (A): Carotenoids Extracted from Thraustochytrium sp. CHN-1, (B): Synthetic
Astaxanthin.
(1);
-carotene; (2)+ echinenone; (3); canthaxanthin; (4), (6), (7); unknown; (5); phenocoxanthin; (8); (3S,30 S)-trans astaxanthin; (9);
(3R,30 S)-trans astaxanthin; (10); (3R,3R0 )-trans astaxanthin; (11); (3S,30 S)-cis astaxanthin; (12): (3R,30 S)-cis astaxanthin; (13): (3R,3R0 )-cis
astaxanthin. (A) HMBC and NOESY Correlations in Peak 8.

data are reported as the mean.
Freeze-dried samples (approximately 50 mg) were
extracted with 100% acetone, then carotenoids were
analyzed by HPLC (Shimazu type LC-8A). A chirality
HPLC column was used as a Pirkle-covalent L-
leucine column, 5 m particle size, 25 cm  4.6 mm
(Regis Chemical Co., Morton Grove, Illinois, U.S.A).
The HPLC mobile phase was conducted with
hexane-tetrahydrofuran (THF)-2-propanol-triethylamine
(77:17:3:3). The ow rate was 1 ml/min, and the
monitoring wavelength 460 nm. Typical chirality col-
umn HPLC chromatographic data were obtained from
the acetone extract of Thraustochytrium sp. CHN-1(a)
and synthetic astaxanthin (b); (Homann-La Roche,
Inc.). These data are shown in Fig. 1. Thraustochytrium
sp. CHN-1 clearly indicates the presence of 10 dominant
carotenoid compounds in the acetone extracts. Peaks 1,
2, 3, and 5 were identied as
-carotene, echinenone,
canthaxanthin, and phenocoxanthin because they had
identical adsorption spectra, retention times, mass
spectra from LC-MS, and spectra of PDA.3)
Six main astaxanthins were separated from synthetic
astaxanthin. This HPLC chromatographic pattern of
synthetic astaxanthin showed agreement with results
1596 Y. YAMAOKA et al.
9)
reported by Turujima et al. Synthetic all-trans and cis-
astaxanthin consist of a racemic mixture of the two
enanti-isomers and the meso form.9) Peaks 8, 10, and 11
in the chromatogram of Thraustochytrium sp. CHN-1(a)
were identied as (3S,3S0 )-trans-astaxanthin, (3R,3R0 )-
trans-astaxanthin, and (3S,3S0 )-cis-astaxanthin by agree-
ment with the retention time of synthetic astaxanthin.
A astaxanthin (Peak 8) had HREIMS m=z: 596.4
(calculated for C40 H52 O4 : 596.4) (
 0:9 mmu,

1:6 ppm); EIMS m=z (rel. int.): 596 (30), 496 (50), 339
(100); UV max (CH2 Cl2 ) nm ("): 486.5 (86.700), 298.5
(" 15,000), 250.5 (16,400); CD (CH2 Cl2 ) (
"), 385
(94.4), 320 (18.0), 277 (9.2), 248 (12.3), 229
(11.7) The extract structure of astaxanthin was
inferred from detailed analysis of 1 H- and 13 C NMR
data together with HMBC and NOESY (Fig. 1). To-
gether with CD spectrum and NMR analysis results, we
concluded that Thraustochytrium sp. CHN-1 produces
(3S,3S0 )-all trans-astaxanthin. The red yeast Phaa
rhodozyma4) contains high concentrations of (3R,3R0 )-all
trans-astaxanthin diesters. Astaxanthin in Haematococ-
cus pluvialis was composed of (3S,3S0 )-all trans,
(3R,3R0 )-all-trans, (3S,3S0 )-13-cis-astaxanthin, (3S,3S0 )-
9-cis-astaxanthin, and astaxanthin esters (monoester
7080% and diester 1020%).10) A astaxanthin fatty
acid ester, however could not be separated from
Thraustochytrium sp. CHN-1. Natural carotenoids are
generally present in the most stable structural form, all-
trans, in which all of the double bonds are in the trans
conguration. Cis-isomers of carotenoid pigments are
less stable in light and may oxidize more rapidly than
the trans compounds.11)
Next we examined the eects of intense uorescent
light (1,500 lux), and red, blue, and near-infrared LED
light on growth. Figure 2 shows that change in the
growth curve was observed in dark and light conditions.
The strain grew even in a medium without light, but cell
growth obtained in dark conditions was about half that
in uorescent light conditions. Biomass yields from 15 d
incubation with the three LEDs were 22.4 g dry cell/l.
The biomass order of Thraustochytrium sp. CHN-1
incubated for 15 d in various light conditions was
Fig. 2. Growth Curve of Thraustochytrium sp. CHN-1 under a
Various LED Light Conditions.
; Fluorescent; ; Red; ; Blue; ; Near-intrared; ; Dark.
Fig. 3. Biomass and Carotenoids Accumulation from Thraustochy-
trium sp. CHN-1 under a Various LED Light Conditions.
; Biomass; ; Astaxanthin; ; Phenocoxanthin; ; Canthax-
anthin.
uorescent > red LEDs > blue LEDs > near-infrared
LEDs > dark. This result suggests that light conditions
aected Thraustochytrium growth. Cultures of Thraus-
tochytrium sp. CHN-1 grown under low intensity
uorescent light (1,500 lux) were orange and red in
color. Carotenoids in Thraustochytrium sp. CHN-1 were
separated and indicated as astaxanthin, phenocoxanthin,
canthaxanthin, echinenone, and
-carotene.3) Figure 3
shows biomass and carotenoid accumulation from
Thraustochytrium sp. CHN-1 in various LED light
conditions. The order of carotenoid production of
Thraustochytrium sp. CHN-1 incubated for 15 d under
various light conditions was blue LEDs > uorescent >
red LEDs > dark > near-infrared LEDs. Carotenoid
yields under the blue LED condition were ve times
those under the dark condition. Blue LEDs induced
accumulation of more carotenoids in the cells. The order
of carotenoid composition of Thraustochytrium sp.
CHN-1 incubated for 15 d under the blue LED condition
was astaxanthin > phenocoxanthin > canthaxanthin.
These results demonstrate that astaxanthin production by
Thraustochytrium sp. CHN-1 with blue LEDs exceeds
that in uorescent light. Blue light activates various
metabolite developments (carotenoid synthesis, etc.) and
behavioral processes both in plants and in prokaryotic
and eukaryotic micro-organisms.12) More detailed ex-
amination of blue LED the eects on the mechanisms of
astaxanthin production will be necessary in the future
study.
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