Introduction
Astaxanthin (3,3¢-dihydroxy-,¢-carotene-4,4¢-dione)
is a useful carotenoid for inducing pigment formation
in fish during aquaculture. Moreover, astaxanthin
possesses a higher antioxidative activity than -carotene
and ␣-tocopherol, so that astaxanthin is attractive as a
powerful antioxidative reagent (Miki, 1991). The yeast,
Phaffia rhodozyma, is used in the industrial production
of astaxanthin (An et al., 1996).
Recently, it has reported that some of the precursors
such as monoterpenes (Meyer et al., 1994) and meval-
onate (Calo et al. 1995) are effective for the astaxanthin
production by P. rhodozyma on the glucose medium.
Since these substances are costly, they are impractical
for industrial-scale production. In the present study, we
investigated the effect of various carbon sources on the
astaxanthin production by P. rhodozyma, to search for
the carbon source which was less expensive and more
effective for astaxanthin production. Then, we devel-
oped a two-stage fed-batch culture system, in which the
cell growth was optimized by glucose feeding, and the
astaxanthin production was enhanced by the supply of
the selected substrate (ethanol).
Materials and methods
Microorganism and medium
P. rhodozyma ATCC 24202 was grown on the basal
medium containing (per liter): 20 g glucose, 5.0 g
(NH
4
)
2
SO
4
, 1.0 g KH
2
PO
4
, 0.5 g MgSO
4
.
7H
2
O, 0.1 g
CaCl
2
.
2H
2
O, 1.0 g yeast extract, 0.1 ml A-nol (Able
Co., Tokyo, Japan) as antifoam and 20 g potassium
biphthalate as a buffer (only flask culture), and pH
adjusted to 4.5.
Flask culture
The preculture broth (2 ml) was inoculated into 100 ml
Erlenmeyer flask containing 18 ml of the basal medium
and cultivated with shaking at 140 rpm at 20°C for
96 h.
Two-stage fed-batch cultures with pH-stat and
DO-stat
The preculture broth (200 ml) was inoculated into 5 l
jar-fermenter containing 1.8 l of the basal medium.
When the residual glucose concentration decreased to
5 g/l, glucose (500 g/l) was provided in response to pH
decrease as described by Nishio et al. (1977). The
pH was controlled at 4.4–4.5 by feeding of 2 M
NH
4
OH solution. The nutrients except for glucose and
NH
4
+
were intermittently supplied at an appropriate
time. Dissolved oxygen concentration (DO) was
controlled at above 5 mg/l by increasing the agitation
speed. Aeration speed and the initial agitation speed
were set at 1.0 vvm and 500 rpm, respectively.
Temperature was controlled at 20°C.
When high cell concentration was attained in the
glucose fed-batch culture, ethanol feeding was
performed by the DO-stat method (Yano et al. 1991).
When the on-line monitored DO level was increased
due to the substrate depletion, a certain amount of
ethanol (purity: 99.5%) was supplied until the DO level
dropped to 5.0–5.2 mg/l.
Analyses
The growth, glucose and astaxanthin were analyzed as
previously described (Yamane et al. 1996). Ethanol was
monitored by gas chromatography (Nishio et al. 1982).
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Biotechnology Letters, Vol 19, No 11, November 1997, pp. 1109–1111
Biotechnology Letters · Vol 19 · No 11 · 1997
1109
Astaxanthin production by
Phaffia
rhodozyma
enhanced in fed-batch
culture with glucose and ethanol feeding
Y. Yamane, K. Higashida, Y. Nakashimada, T. Kakizono and N. Nishio*
Department of Fermentation Technology, Faculty of Engineering, Hiroshima University, Kagamiyama,
Higashi-Hiroshima 739, Japan
Of various carbon sources, examined for the cultivation of
Phaffia rhodozyma
, ethanol enhanced the astaxanthin
content but severely decreased growth. Therefore, high cell mass was obtained by glucose fed-batch culture with
pH-stat, and the ethanol feeding was performed based on DO-stat. As a result of this two-stage fed-batch cultiva-
tion, 30 g dry cells per liter were obtained, and the astaxanthin content reached 0.72 mg/g, which was 2.2-fold higher
than that without ethanol feeding.
© 1997 Chapman & Hall
J BL,1109-1111,904 Yam 20/10/97 8:11 am Page 1109