APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1997, p. 4471–4478 Vol. 63, No.

11
0099-2240/97/$04.0010
Copyright © 1997, American Society for Microbiology

Influence of Oxygen and Glucose on Primary Metabolism and

Astaxanthin Production by Phaffia rhodozyma in Batch and
Fed-Batch Cultures: Kinetic and Stoichiometric Analysis
YU-ICHI YAMANE, KATSUYA HIGASHIDA, YUTAKA NAKASHIMADA,
TOSHIHIDE KAKIZONO, AND NAOMICHI NISHIO*
Department of Fermentation Technology, Faculty of Engineering, Hiroshima University,
Kagamiyama 1-4-1, Higashi-Hiroshima 739, Japan
Received 24 March 1997/Accepted 17 August 1997

The influence of the oxygen and glucose supply on primary metabolism (fermentation, respiration, and
anabolism) and astaxanthin production in the yeast Phaffia rhodozyma was investigated. When P. rhodozyma
grew under fermentative conditions with limited oxygen or high concentrations of glucose, the astaxanthin
production rate decreased remarkably. On the other hand, when the yeast grew under aerobic conditions, the
astaxanthin production rate increased with increasing oxygen uptake. A kinetic analysis showed that the
respiration rate correlated positively with the astaxanthin production rate, whereas there was a negative cor-
relation with the ethanol production rate. The influence of glucose concentration at a fixed nitrogen concen-
tration with a high level of oxygen was then investigated. The results showed that astaxanthin production was
enhanced by an initial high carbon/nitrogen ratio (C/N ratio) present in the medium, but cell growth was in-
hibited by a high glucose concentration. A stoichiometric analysis suggested that astaxanthin production was
enhanced by decreasing the amount of NADPH required for anabolism, which could be achieved by the re-
pression of protein biosynthesis with a high C/N ratio. Based on these results, we performed a two-stage fed-
batch culture, in which cell growth was enhanced by a low C/N ratio in the first stage and astaxanthin
production was enhanced by a high C/N ratio in the second stage. In this culture system, the highest astaxan-
thin production, 16.0 mg per liter, was obtained.

Astaxanthin (3,39-dihydroxy-b,b9-carotene-4,49-dione) is an stress, and they have speculated that astaxanthin plays a role as
abundant carotenoid pigment in some marine animals such as an active oxygen scavenger in the cell. In addition, Schroeder
salmonids and crustaceans. Since these animals cannot synthe- and Johnson (30) have reported that carotenoid biosynthesis is
size astaxanthin, this carotenoid has been added to the feed of regulated by singlet oxygen and peroxyl radicals in the P. rho-
the aquacultivated salmonid, trout, and prawn to improve their dozyma cell. Based on these two studies, it could be considered
color quality (15). Moreover this carotenoid possesses a higher that oxygen might play an important role in astaxanthin bio-
antioxidant activity than b-carotene and a-tocopherol (32). synthesis. On the other hand, Arnezeder and Hampel (4, 5)
Therefore, astaxanthin has attained a commercial interest not have reported that the production of ergosterol, which, like
only as a pigmentation source for fish aquaculture but also as astaxanthin, is synthesized via a mevalonate pathway (10), is
a powerful antioxidative reagent (18). Astaxanthin has been
enhanced by nitrogen- or phosphate-limited culture of Saccha-
produced mainly by the chemical synthetic method on an in-
romyces cerevisiae. Therefore, it may be that astaxanthin pro-
dustrial scale. In recent years, however, the use of chemical
synthetic compounds as food additives has been strictly regu- duction could be enhanced under such nutrient-limited condi-
lated. Since a few microorganisms are capable of synthesizing tions, especially with a high carbon/nitrogen ratio (C/N ratio)
astaxanthin, the establishment of a natural astaxanthin source in the medium. Kakizono et al. (17) have reported that the
by these microorganisms is now required. production of astaxanthin by H. pluvialis is enhanced by a high
An astaxanthin-producing yeast, Phaffia rhodozyma, has C/N ratio, which in their case was caused by an excess supply of
some advantageous properties: (i) it synthesizes astaxanthin as acetate (carbon source). Many workers have reported that
a principal carotenoid, (ii) it does not require light for its primary metabolism, such as anabolism and catabolism (fer-
growth and pigmentation, (iii) it can utilize many kinds of mentation and respiration), varies with the excess supply of
saccharides under both aerobic and anaerobic conditions, and oxygen or carbon source (8, 13, 19, 21, 24, 25, 31). Therefore,
(iv) it can grow at a rate of 0.10 to 0.15 h21 (3, 14, 16). a change in primary metabolism might be a trigger for high
Therefore, P. rhodozyma is attractive for commercial astaxan- astaxanthin accumulation.
thin production, and many studies have reported improve- In the present study, we investigated the influence of the
ments in astaxanthin production (2, 12, 20, 22, 23, 29). oxygen and glucose supply on primary metabolism and astax-
Recently, Kobayashi et al. (18) have reported that astaxan- anthin production by P. rhodozyma. First, the influences of
thin production by Haematococcus pluvialis, an astaxanthin- fermentative conditions and oxygen supply on astaxanthin pro-
producing microorganism, is much enhanced by oxidative duction were investigated by using a kinetic analysis. Second,
the influence of the glucose supply at a fixed nitrogen concen-
tration with high levels of oxygen was investigated. Third,
* Corresponding author. Mailing address: Department of Fermen-
tation Technology, Faculty of Engineering, Hiroshima University, based on the results from the batch culture, we operated a
Kagamiyama 1-4-1, Higashi-Hiroshima 739, Japan. Phone: 81-824-24- two-stage fed-batch culture with a control C/N ratio in the feed
7760. Fax: 81-824-22-7191. E-mail: nnishio@ipc.hiroshima-u.ac.jp. medium. Finally, we analyzed the correlation between primary

4471
4472 YAMANE ET AL. APPL. ENVIRON. MICROBIOL.

metabolism and astaxanthin production, based on a stoichio- gas, RO2-out is the percent oxygen in the exhaust gas, RCO2-in is the percent carbon
metric analysis. dioxide in the inlet gas, and RCO2-out is the percent carbon dioxide in the exhaust
gas. The oxygen uptake and carbon dioxide evolution rates were calculated as
follows:
MATERIALS AND METHODS
dO2 /dt 5 @Fin~RO2-in/100! 2 Fout~RO2-out/100!#/22.4V
Microorganism and medium. P. rhodozyma ATCC 24202 was used. This yeast
was maintained at 4°C on an agar slant containing (per liter) 10 g of glucose, 5.0 g dCO2 /dt 5 @Fin~RCO2-out/100! 2 Fout~RCO2-in/100!#/22.4V
of Bacto Peptone, 3.0 g of malt extract, 3.0 g of yeast extract, and 20 g of agar.
YM medium contained (per liter) 10 g of glucose, 5.0 g of Bacto Peptone, 3.0 g where dO2/dt is the oxygen uptake rate (moles per liter per hour), dCO2/dt is the
of malt extract, and 3.0 g of yeast extract and was adjusted to pH 5.0. The basal carbon dioxide evolution rate (moles per liter per hour), and V is the culture
medium contained (per liter) 20 g of glucose, 5.0 g of (NH4)2SO4, 1.0 g of volume (liters).
KH2PO4, 0.5 g of MgSO4 z 7H2O, 0.1 g of CaCl2 z 2H2O, 1.0 g of yeast extract, Calculation of carbon recovery. In order to estimate the material balance
0.1 ml of antifoam reagent (A-nol; Able, Tokyo, Japan), and 20 g of potassium between (i) glucose consumption and (ii) cell growth and carbon dioxide evolu-
biphthalate (used only for preculture and flask batch culture as a buffer) and was tion, carbon recovery was calculated as follows:
adjusted to pH 4.5. Bacto Peptone, malt extract, yeast extract, and agar were
RC 5 ~gXDX 1 gCO2DCO2)/gSDS (1)
obtained from Difco Laboratories (Detroit, Mich.).
The starter culture broth (4 ml; cultivated with 10 ml of YM medium in a 50-ml where Rc is the carbon recovery (dimensionless), DCO2 is the carbon dioxide
test tube for 72 h) was inoculated into a 200-ml Erlenmeyer flask containing 36 evolution (moles per liter), DS is the glucose consumption (grams per liter), DX
ml of the basal medium, and a preculture was carried out for 48 h. Both the is the final cell concentration (grams per liter), gCO2 is the carbon content in
starter culture and the preculture were performed with shaking at 140 rpm on a carbon dioxide (12 g per mol), gS is the carbon content in glucose (0.40 g per g),
rotary shaker at 20°C. and gX is the carbon content in the dry cell (grams per gram; defined as 0.50 g
Batch culture. In the flask batch culture, the preculture broth (2 ml) was per g).
inoculated into a 100-ml Erlenmeyer flask containing 18 ml of the basal medium. Calculation of specific rates. The maximum specific growth rate, mmax
The flask batch culture was performed with shaking at 140 rpm on a rotary (hours21), was calculated by a linear regression method from the logarithm of
shaker at 20°C. According to the effects of the buffer, the pH was maintained in dry cell concentration versus time during the early exponential growth phase.
the range of 4.3 to 4.5. The specific rates of astaxanthin production (qAxn) (micrograms per gram per
In the fermentor batch culture, the preculture broth (300 ml) was inoculated hour), ethanol production (qE) (milligrams per gram per hour), and oxygen
into a 5-liter jar fermentor (KMJ-5B; Mitsuwa, Osaka, Japan) containing 2.7 uptake (qO2) (millimoles per gram per hour) during the early exponential growth
liters of the basal medium. The temperature was controlled at 20°C. The pH was phase were calculated as follows: qAxn 5 (DAxnexp/DXexp)mmax, qE 5 (DEexp/
monitored with a pH electrode (Toa Electronics, Tokyo, Japan) and adjusted at DXexp)mmax, and qO2 5 (DO2exp/DXexp)mmax, where DAxnexp, DEexp, DO2exp, and
4.4 to 4.5 with a pH controller (MPH-3C; Mitsuwa) by adding a 2 M NaOH DXexp are astaxanthin production (micrograms per liter), ethanol production
solution through a peristaltic pump (NP-3NS; Tokyo Rikakikai, Tokyo, Japan). (milligrams per liter), oxygen uptake (millimoles per liter), and cell growth
The dissolved oxygen (DO) concentration was monitored with a DO electrode (grams per liter), respectively, during the early exponential growth phase.
(Toa Electronics) and controlled with a DO controller (MDO-3C; Mitsuwa) by Analyses. The acetone extraction method, as described by Okagbue and Lewis
changing the aeration or agitation speed. In the batch culture, the DO concen- (28), was used to measure the total carotenoid concentration. Because the ratio
tration was maintained at 0.0, 2.0, 5.0, and 8.0 mg per liter, which corresponded of astaxanthin to total carotenoids produced was more than 90% in P.
to 0.0, 23, 57, and 90% of the saturation value (8.84 mg per liter at 20°C in rhodozyma, this method was employed, with slight modifications, to measure the
distilled water), respectively. The DO concentration could be maintained at the astaxanthin concentration. The procedures were as follows. The cells in 10 ml of
desired level in the range of 60.1 mg per liter. the culture broth were centrifuged (5,000 3 g, 10 min) and maintained at 280°C
Two-stage fed-batch culture. The preculture broth (200 ml) was inoculated for 2 h. After addition of 2 ml of 2.5 M HCl, the cells were heated for 2.5 min
into a 5-liter jar fermentor containing 1.8 liters of the basal medium in which the in a boiling water bath, quickly cooled, and centrifuged (5,000 3 g, 10 min). After
(NH4)2SO4 concentration was reduced to 0.25 g per liter. The first stage of the the cells were washed twice with deionized water, 6 ml of acetone was added to
fed-batch culture was started when the residual glucose concentration in the the cells, and the samples were maintained at 4°C for 1.5 h. An excess amount of
batch culture fell to about 5.0 g per liter. To prevent precipitate formation, the Na2SO4 powder was then added to remove the water, and the cells were main-
phosphorous source and minerals were fed separately. Therefore, three feeding tained at 4°C for 30 min. After centrifugation (5,000 3 g, 10 min), the absorbance
solutions were prepared: a glucose solution of 500 g per liter; a mineral solution at 478 nm was measured with a spectrophotometer (UV-1600; Shimadzu, Kyoto,
consisting of 12.5 g of MgSO4 z 7H2O, 2.5 g of CaCl2 z 2H2O, and 25 g of yeast Japan). The astaxanthin concentration was calculated by using the absorption
extract per liter; and a KH2PO4 solution of 25 g per liter. The feedings of these coefficient A1% 5 2,100 (2).
three solutions were set at the same rate (180 ml per h) and were performed The cell concentration was measured by optical density at 600 nm or by dry cell
simultaneously in response to the pH decrease (26) by using three peristaltic weight, after filtration on cellulose nitrate (0.45-mm pore size; Advantec Toyo,
pumps connected to a pH controller. In the batch culture phase and the first Tokyo, Japan) and drying at 105°C for 24 h. The ratio of the optical density to dry
stage of the fed-batch culture, pH control was performed with a 2 M ammonium cell weight (grams per liter) was 2.50 6 0.10.
solution to supply a nitrogen source. The feed rate of the ammonium solution The protein content in the cell was measured by a modified biuret method (1,
was set at 90 ml per h, which was half the rate used for the glucose solution. The 34) as follows. Ten milliliters of the culture broth was centrifuged (5,000 3 g, 10
DO concentration was controlled at 5.0 mg per liter by changing the agitation min) and washed twice with deionized water. Then 3 ml of 1 M NaOH was added
speed. and boiled for 10 min. After the broth was quickly cooled CuSO4 was added to
In the second stage of the fed-batch culture, the three pumps for the glucose, give a final concentration of 25 mM. After 5 min at room temperature (25°C), the
mineral, and phosphate solutions were connected to the DO controller. The mixture was centrifuged (13,000 3 g, 2 min). The optical density at 550 nm was
pump for the ammonium solution was also connected to the DO controller. In then measured by using bovine serum albumin (Sigma Chemical Co., St. Louis,
this stage, the feedings of these four solutions were performed in response to an Mo.) as a standard.
abrupt increase in the DO concentration, since this increase could be observed Glucose was measured by the glucose oxidase method (Diacolor-GC; Toyobo,
when the glucose in the culture medium was completely consumed (35). There- Osaka, Japan). Ethanol was measured by gas chromatography (GC-8A; Shi-
fore, the feedings of these solutions were performed when the DO concentration madzu), as described by Nishio et al. (27). Formate, acetate, propionate, and
exceeded 6.0 mg per liter. The feed rates for the glucose, mineral, and phosphate glycerol were measured by high-performance liquid chromatography (TRI
solutions were set at the same values as in the first stage, whereas the feed rate ROTAR-V; JASCO, Tokyo, Japan) with a Shodex Ionpak S-801 column (Showa
of the ammonium solution was set at 22.5 ml per h, which was a quarter of that Denco, Tokyo, Japan) and a UV spectrophotometer (UVIDEC-100-V; JASCO),
used in the first stage. In order to maintain the DO concentration at 5.0 mg per as described by Fukuzaki et al. (9). The ammonium concentration was measured
liter, the agitation speed was increased only when the DO concentration fell by the indophenol method (11).
below 5.0 mg per liter. pH control was performed with a 2 M NaOH solution. All of the analyses were carried out for duplicate or triplicate cultures, and the
Throughout the period of the two-stage fed-batch culture, the temperature average values are reported.
and pH were controlled at 20°C and 4.4 to 4.5, respectively, and the aeration
speed was set at 1.0 volume of air per volume of liquid per min.
Calculation of oxygen uptake rate and carbon dioxide evolution rate. The RESULTS
partial pressures of oxygen and carbon dioxide in the exhaust gas from the
fermentor were monitored with an infrared carbon dioxide and thermomagnetic Effect of fermentative conditions on growth and astaxanthin
oxygen analyzer (model EX-1562-1; Able, Tokyo, Japan). The flow rate of the
exhaust gas was calculated as follows: production. In general, yeasts ferment a sugar and produce
ethanol under oxygen-limiting conditions (Pasteur effect). In
Fout 5 [(100 2 RO2-in 2 RCO2-in)/(100 2 RO2-out 2 RCO2-out)]Fin addition, yeasts also ferment a sugar at high sugar concentra-
where Fin is the aeration rate into the fermentor (liters per hour), Fout is the flow tions, even under aerobic conditions (Crabtree effect). There-
rate of the exhaust gas (liters per hour), RO2-in is the percent oxygen in the inlet fore, in order to examine the effects of the fermentative con-
VOL. 63, 1997 EFFECT OF OXYGEN AND GLUCOSE ON ASTAXANTHIN PRODUCTION 4473

TABLE 1. Effect of fermentative conditions on growth of and astaxanthin production by P. rhodozyma in flask culturea
Culture vol DAxn/DXk
Glucose mmaxb qAxnc qE d DXf DSg YX/Sh DEi DAxnj
(ml/100-ml Dte (h) (mg/g [dry wt]
(g/liter) (h21) (mg/g/h) (mg/g/h) (g/liter) (g/liter) (g/g) (g/liter) (mg/liter)
flask) of cells)

20 20 0.14 6 0.01 39.1 6 4.3 56 5.93 6 0.14 20.0 6 0.0 0.30 NDl 1.66 6 0.15 0.28
40 0.090 6 0.010 12.2 6 1.1 66.0 6 3.8 48 4.75 6 0.18 18.2 6 0.7 0.26 3.89 6 0.11 0.72 6 0.05 0.15
60 0.052 6 0.004 1.53 6 0.22 94.2 6 5.4 80 2.60 6 0.11 17.9 6 0.7 0.14 4.90 6 0.53 0.075 6 0.005 0.029
40 20 0.10 6 0.01 21.0 6 1.6 48.8 6 2.8 81 7.17 6 0.30 40.0 6 0.0 0.18 3.42 6 0.19 1.48 6 0.09 0.21
60 20 0.084 6 0.005 16.0 6 1.7 44.3 6 2.7 96 9.90 6 0.72 58.0 6 2.4 0.17 5.47 6 0.49 2.01 6 0.29 0.20
80 20 0.049 6 0.006 5.02 6 0.38 21.1 6 1.3 168 8.85 6 0.23 70.0 6 0.6 0.13 6.21 6 0.23 1.58 6 0.10 0.18
a
All experimental data are expressed as means and standard deviations derived from triplicate experiments. All cultivations were carried out until no more glucose
was consumed.
b
Maximum specific growth rate.
c
Specific astaxanthin production rate during early exponential phase.
d
Specific ethanol production rate during early exponential phase.
e
Culture time.
f
Final cell concentration.
g
Glucose consumption.
h
Growth yield for glucose consumption (DX/DS). These values were calculated from the means of the experimental data.
i
Final ethanol concentration.
j
Final astaxanthin concentration.
k
Final specific astaxanthin concentration. These values were calculated from the means of the experimental data.
l
ND, not detected.

ditions on growth and astaxanthin production, we performed Effect of oxygen supply on growth and astaxanthin produc-
two series of experiments. First, to investigate the Pasteur tion. In order to avoid the decrease in astaxanthin production
effect, batch cultures were performed with 20 g of glucose per due to the Pasteur effect, the DO concentration must be main-
liter in a 100-ml Erlenmeyer flask with culture volumes of 20, tained at an appropriate level. To investigate the effects of
40, and 60 ml. The results are summarized in Table 1. Because varying the oxygen supply, batch cultures were performed in
no ethanol was produced with a 20-ml culture volume, this the fermentor with the basal medium under DO control at 0.0,
culture was considered to be grown under aerobic conditions. 2.0, 5.0, and 8.0 mg per liter. As shown in Table 2, mmax was
With 40- and 60-ml culture volumes, however, ethanol was maintained at a high level (.0.18 h21) at 5.0 and 8.0 mg of DO
produced. The final ethanol concentration (DE) (grams per per liter but decreased at less than 2.0 mg of DO per liter.
liter) increased, and the maximum specific growth rate (mmax) DAxn was enhanced remarkably at 2.0 mg per liter, whereas
(hours21), the final astaxanthin concentration (DAxn) (milli- there was no significant difference at a DO concentration
grams per liter), and the specific astaxanthin concentration higher than 2.0 mg per liter. From these results, the most
(DAxn/DX) (milligrams per gram) decreased with increasing suitable DO concentration for growth and astaxanthin produc-
culture volume. tion was determined to be 5.0 mg per liter.
Next, to investigate the Crabtree effect, the initial glucose Effect of glucose supply on growth and astaxanthin produc-
concentrations in the batch cultures were increased to 40, 60, tion at high levels of oxygen. To investigate the effects of
and 80 g per liter with a culture volume of 20 ml, since aerobic varying the glucose supply at a fixed nitrogen concentration,
conditions were maintained with a culture volume of 20 ml in batch cultures were performed under DO control at 5.0 mg
the 100-ml Erlenmeyer flask, as mentioned above. These re- per liter in the fermentor. The initial glucose concentrations
sults are also summarized in Table 1. In all of the cultures, ranged from 10 to 120 g per liter, whereas the concentrations
ethanol production was observed. In addition, with an increase
in the initial glucose concentration, DE increased and mmax,
DAxn, and DAxn/DX decreased.
Based on the data in Table 1, a relationship between fer-
mentation and astaxanthin production during the early expo-
nential phase was analyzed by a kinetic procedure. The values
for the specific astaxanthin production rate, qAxn, were plotted
against the values for the specific ethanol production rate, qE.
As shown in Fig. 1, except for one data point, a linear rela-
tionship was observed between qAxn and qE:
qAxn 5 2KAxn/EqE 1 37.8 (2)
where KAxn/E is a kinetic constant which shows the ratio of
astaxanthin production to ethanol production and is estimated
from the slope of the straight line in Fig. 1 to be 0.39 mg of
astaxanthin per mg of ethanol. Equation 2 clearly shows that
astaxanthin production was repressed by the fermentation of P.
rhodozyma. Although a remarkable deviation from equation 2
was observed at 80 g of glucose per liter (Fig. 1), this could be FIG. 1. Relationship between the initial specific astaxanthin production rate
(qAxn) and the specific ethanol production rate (qE) during the early exponential
due to physiological changes in cell metabolism due to a con- phase. The mean values from Table 1 are shown. The line was calculated by the
siderable decrease in mmax and the remarkably long culture least-squares regression method (r 5 0.983), except for the data (F) obtained for
time. 80 g of glucose per liter.
4474 YAMANE ET AL. APPL. ENVIRON. MICROBIOL.

TABLE 2. Effect of oxygen supply on growth of and astaxanthin production by P. rhodozyma in a fermentor under DO controla
DAxn/DXc
DO mmax qAxn qO2b DX YX/Sc DAxn
Dt (h) (mg/g [dry wt]
(mg/liter) (h21) (mg/g/h) (mmol/g/h) (g/liter) (g/g) (mg/liter)
of cells)

0.0 0.10 6 0.01 16.0 6 2.1 1.50 6 0.12 48 4.80 6 0.21 0.24 0.75 6 0.01 0.16
2.0 0.13 6 0.01 43.2 6 5.2 4.16 6 0.23 42 9.40 6 0.05 0.47 3.10 6 0.10 0.33
5.0 0.18 6 0.01 54.0 6 4.3 5.22 6 0.30 36 9.27 6 0.20 0.46 2.79 6 0.01 0.30
8.0 0.19 6 0.01 63.0 6 5.3 5.89 6 0.29 30 10.2 6 0.08 0.51 3.39 6 0.01 0.33
a
All experimental data are expressed as means and variances derived from duplicate experiments. Nomenclature is the same as in Table 1. All cultivations were
carried out in a fermentor with the basal medium containing 20 g of glucose per liter and were continued until glucose was consumed completely.
b
Specific oxygen uptake rate during early exponential phase.
c
Calculated from the means of the experimental data.

of the other medium components were not changed from those Crabtree effect and other inhibitory effects derived from the
in the basal medium. The results are summarized in Table 3. high concentrations of glucose. At 120 g of glucose per liter,
Ethanol production was not detected or was at trace levels however, qAxn decreased remarkably, even if the DO concen-
(,0.3 g per liter) in all cultures, suggesting that the Crabtree tration was maintained at a high level. Since a considerable
effect was repressed by a high oxygen supply. Extracellular decrease in mmax was observed and a remarkably long culture
products such as formate, acetate, propionate, and glycerol time was required at 120 g of glucose per liter (Table 3), this
were not detected in any of the cultures (except for the trace was considered to be due to the physiological changes in cell
amount of ethanol). The maximum specific growth rate de- metabolism stemming from the inhibitory effects of high glu-
creased with increasing initial glucose concentration. This might cose concentrations.
be due to an inhibitory effect of high concentrations of glucose. The results shown in Fig. 2 clearly show that oxygen uptake
The final cell concentration (DX), DAxn, and DAxn/DX in- plays an important role in astaxanthin production. Therefore,
creased with increasing initial glucose concentration, but they the role of oxygen uptake, or respiration, on astaxanthin pro-
decreased at 120 g of glucose per liter. At 80 g of glucose per duction was then analyzed by a kinetic procedure. The values
liter, the highest DAxn (14.8 mg per liter) was obtained in all of qAxn in Tables 2 and 3 were plotted against the values of qO2.
batch cultures. DAxn/DX reached 0.51 mg per g, which was As shown in Fig. 3, a linear relationship between qAxn and qO2,
1.8-fold higher than that at 10 g of glucose per liter. The except for one data point, was obtained:
residual ammonium concentration decreased with increasing
the initial glucose concentration and was not detected at 80 g qAxn 5 KAxn/O2qO2 1 0.23 (3)
of glucose per liter, indicating the presence of an ammonium
deficit. These results suggest that at high DO concentrations, where KAxn/O2 is a kinetic constant representing the ratio of the
astaxanthin production is enhanced by an initial high C/N ratio rate of astaxanthin production to the rate of oxygen uptake,
in the medium. which is estimated from the slope of the straight line in Fig. 3
The effect of the glucose supply on astaxanthin production to be 10.3 mg of astaxanthin per mmol of oxygen. Equation 3
was then analyzed kinetically. The values of qAxn were plotted clearly shows that astaxanthin production was enhanced by an
against the initial glucose concentration (Fig. 2). The values of increase in the respiratory activity of P. rhodozyma. A remark-
qAxn at various glucose levels in the flask cultures (Table 1) able deviation from equation 3 was observed at 120 g of glu-
were also plotted (Fig. 2) to compare them with those values at cose per liter (Fig. 3), which may be due to the reasons men-
high levels of oxygen. In the flask culture, in which it was tioned in the discussion of Fig. 2 above.
difficult to maintain DO at a high level throughout the entire In order to analyze their influences on anabolism, the growth
cultivation period, qAxn decreased remarkably as the initial yield for glucose consumption (YX/S), the ratio of cell growth to
glucose concentration was increased. On the other hand, qAxn nitrogen consumption (DX/DN), the ratio of cell growth to
could be successfully maintained at almost 0.54 mg per g per h oxygen uptake (DX/DO2), the ratio of cell growth to carbon
at 10 to 80 g of glucose per liter under DO control at high dioxide evolution (DX/DCO2), and the ratio of astaxanthin
levels. These results suggest that to maintain high astaxanthin production to glucose consumption (DAxn/DS) were calculated
productivity, high levels of oxygen are necessary to repress the from the data obtained in Table 3 (with the omission of the

TABLE 3. Results for DO-controlled batch cultures with various initial glucose concentrationsa
DAxn/DXc
Glucose mmax qAxn qO2 DX DS Residual Nb DAxn
Dt (h) (mg/g [dry wt]
(g/liter) (h21) (mg/g/h) (mmol/g/h) (g/liter) (g/liter) (g/liter) (mg/liter)
of cells)

10 0.19 6 0.01 55.0 6 0.3 5.50 6 0.20 26 4.56 6 0.34 10 6 0 1.03 6 0.08 1.34 6 0.17 0.29
20 0.18 6 0.00 54.0 6 0.2 5.22 6 0.10 36 9.27 6 0.20 20 6 0 0.69 6 0.04 2.79 6 0.01 0.30
40 0.13 6 0.00 54.6 6 2.1 5.20 6 0.25 46 16.1 6 0.6 40 6 0 0.24 6 0.02 6.74 6 0.35 0.42
80 0.10 6 0.01 51.0 6 3.2 5.00 6 0.30 96 28.8 6 1.7 80 6 0 NDd 14.8 6 0.8 0.51
120 0.078 6 0.005 27.3 6 2.8 6.28 6 0.40 146 22.6 6 1.6 110 6 8 ND 7.80 6 0.80 0.35
a
All values are expressed as means and variances derived from duplicate experiments. All cultivations were performed under DO control at 5.0 mg per liter.
Nomenclature is the same as in Tables 1 and 2.
b
Residual ammonium concentration.
c
Calculated from the means of the experimental data.
d
ND, not detected.
VOL. 63, 1997 EFFECT OF OXYGEN AND GLUCOSE ON ASTAXANTHIN PRODUCTION 4475

FIG. 2. Influence of the initial glucose concentration on the specific astax- FIG. 3. Relationship between the specific astaxanthin production rate (qAxn)
anthin production rate (qAxn) during the early exponential phase. Symbols: E, and the initial specific oxygen uptake rate (qO2) during the early exponential
mean values obtained in batch cultures under DO control at 5.0 mg per liter phase. The mean values from Tables 2 and 3 are shown. The line was calculated
(Table 3); F, mean values obtained in flask batch cultures (Table 1). by the least-squares regression method (r 5 0.999), except for the data (F)
obtained for 120 g of glucose per liter.

data point at 120 g of glucose per liter). As shown in Table 4, M). Since no pH-altering extracellular products were detected
YX/S, DX/DO2, and DX/DCO2 decreased with increasing initial in the aerobic culture of P. rhodozyma, as mentioned above,
glucose concentration, whereas DX/DN increased. The varia- the decrease in pH was considered to be primarily due to the
tions in YX/S, DX/DO2, DX/DCO2, and DX/DN suggest that ammonium consumption. In such a case, exponential cell
changes in anabolism occur as a result of variations in the C/N growth could easily be maintained by the simultaneous feeding
ratio due to the varying glucose supply. On the other hand, of a glucose solution and an ammonium solution (for pH
DAxn/DS increased with increasing initial glucose concentra- control), which would respond to decreases in pH (26). In the
tion. Thus, it was considered that an increase in astaxanthin first stage, therefore, the glucose solution was fed simulta-
production might be accompanied by a change in anabolism. neously with an ammonium solution, for pH control.
To examine the material balance between (i) glucose con- In the second stage of the fed-batch culture, it was necessary
sumption and (ii) cell growth and carbon dioxide evolution, the to enhance astaxanthin production by a medium feed with a
carbon recovery, RC was calculated. Larsson et al. (19) have high C/N ratio. Therefore, the feed rate for the ammonium
reported that the carbon content in the S. cerevisiae cell was solution was set at 12.5% of that for the glucose solution, and
almost constant at 0.50 g of carbon per g (dry weight) of cells the medium feed was performed with the C/N ratio at 11.2 mol
at each dilution rate in chemostat cultures under carbon-, of glucose per mol of ammonium. It is well known that an
nitrogen-, or carbon- and nitrogen-limiting conditions. In the abrupt increase in DO concentration is observed in an aerobic
present study, therefore, it was speculated that the carbon culture when the carbon source has been consumed (35). In
content in the cell, gX in equation 1 should be 0.50 g per g. As the second stage, therefore, the glucose and ammonium solu-
RC could be regarded as almost 1.00 (Table 4), the glucose tions were fed simultaneously in response to an increase in DO
consumed would primarily be converted to cell materials and concentration.
carbon dioxide in all cultures. Typical time profiles for the two-stage fed-batch culture are
Two-stage fed-batch culture. As shown in Table 3, cell shown in Fig. 4, and the results are summarized in Table 5.
growth was inhibited by high concentrations of glucose. To As shown in Table 5, a high cell concentration, 20.4 g per liter,
maintain the glucose concentration at a low level, therefore, a was obtained in a relatively short period of time (40 h), giving
fed-batch culture is suitable for P. rhodozyma cultivation. As mmax 5 0.12 h21 in the first stage. In the second stage, a
shown in Tables 3 and 4, the astaxanthin production was en- relatively high level of astaxanthin production, 10.2 mg per
hanced by a high initial C/N ratio in the medium, whereas a
lower C/N ratio was suitable for cell growth. Thus, to simulta-
neously obtain high cell mass and high astaxanthin production, TABLE 4. Calculated results from the experimental data
the cultivation period should be separated into two stages: the in the DO-controlled batch culturesa
growth stage (first stage), in which the medium feed maintains
a low C/N ratio, and the astaxanthin production stage (second Glucose YX/S DX/DNb DX/DO2c DX/DCO2d DAxn/DSe
RC f
stage), in which the medium feed maintains a high C/N ratio to (g/liter) (g/g) (g/g) (g/mol) (g/mol) (mg/g)
enhance astaxanthin production. 10 0.46 13.8 35.1 30.4 0.13 1.09
In the first stage of the fed-batch culture, efficient cell 20 0.46 13.8 34.3 32.0 0.14 1.01
growth must be attained. As shown in Table 3, the highest mmax 40 0.40 14.4 25.2 23.7 0.17 0.93
was obtained in the batch culture at 10 g of glucose per liter. In 80 0.36 21.2 19.9 19.3 0.19 1.01
this culture, the ratio of consumption of glucose to consump- a
All values were calculated from the means of the experimental data shown in
tion of ammonium during exponential growth was calculated Table 3.
as 2.80 mol of glucose per mol of ammonium. In order to b
Ratio of final cell concentration to ammonium consumption.
c
perform the medium feed with the C/N ratio at 2.80 mol of Ratio of final cell concentration to oxygen uptake.
d
Ratio of final cell concentration to carbon dioxide evolution.
glucose per mol of ammonium, the feed rate of the glucose e
Ratio of final astaxanthin concentration to glucose consumption.
solution (500 g per liter, which is almost equal to 2.80 mol per f
Carbon recovery for glucose consumption against final cell concentration and
liter) was set at a value twice that of the ammonium solution (2 carbon dioxide evolution.
4476 YAMANE ET AL. APPL. ENVIRON. MICROBIOL.

FIG. 4. Results for two-stage fed-batch culture. The thick and thin arrows show the starts of medium feeding with pH and DO control, respectively. Symbols: F,
cell concentration; h, glucose concentration; E, astaxanthin concentration; Ç, astaxanthin content.

liter, was obtained. DAxn/DX in the second stage was ca. 3-fold stage fed-batch cultures used in the present study are very effec-
higher than that in the first stage and 1.5-fold higher than that tive for maximizing P. rhodozyma’s production of astaxanthin.
in the DO-controlled batch culture at 80 g of glucose per liter
(Table 3), indicating that the astaxanthin production was en- DISCUSSION
hanced much more in the second stage than in the first stage.
As a result of the two-stage fed-batch culture, DX and DAxn The results in the present study indicate that astaxanthin
reached 33.6 g per liter and 16.0 mg per liter, respectively, production by P. rhodozyma is affected by fermentation, respi-
which were the highest values for all of the cultivations per- ration, and anabolism. Two questions then arise: (i) why astax-
formed in the present study. These results suggest that the two- anthin production is enhanced by respiration and repressed by

TABLE 5. Results for two-stage fed-batch culturea

mmax DX DXtb DStc DAxn DAxntd DAxn/DX (mg/g
Stage Dt (h)
(h21) (g/liter) (g) (g) (mg/liter) (mg) [dry wt] of cells)

First (growth) 0.12 40 20.4 43.2 104.3 5.80 12.3 0.28

Second (astaxanthin production) 0.03 74 13.2 29.9 166.9 10.2 23.3 0.78

First 1 second 114 33.6 73.1 271.2 16.0 35.6 0.48

a
Results are from a single experiment. Nomenclature is the same as in Tables 1 and 2.
b
Total cell production.
c
Total glucose consumption.
d
Total astaxanthin production.
VOL. 63, 1997 EFFECT OF OXYGEN AND GLUCOSE ON ASTAXANTHIN PRODUCTION 4477

fermentation and (ii) why astaxanthin production is enhanced TABLE 6. Estimated results for the primary metabolism
by a higher initial C/N ratio at high levels of oxygen. To answer of P. rhodozyma in batch culturea
these two questions, we analyzed the experimental data by a Glucose N/C c DNADPHd
stoichiometric procedure. Formula Mol wtb
(g/liter) (g/g) (mmol/g)
To answer the first question, we will discuss the NADH
balance. It is known that under oxygen-limiting conditions, 10 CH1.74O0.52N0.10 24.7 0.057 8.1
NADH accumulates due to a decrease in the efficiency of 20 CH1.71O0.53N0.10 24.5 0.057 7.3
40 CH1.44O0.49N0.09 23.7 0.053 4.0
oxidative phosphorylation (33). Therefore, to maintain the 80 CH1.44O0.56N0.06 24.5 0.034 2.9
NADH balance in cell, it is necessary that the excess NADH be
reoxidized, and it would be decreased by repressing a biosyn- a
All values were estimated from the values shown in Table 4.
b
thetic pathway which produces a large amount of NADH. Estimated molecular weight of cell.
c
Estimated nitrogen content in cell.
Furthermore, Johnson and Schroeder (16) have described a d
Estimated NADPH consumption per gram (dry weight) of cells formed (see
reaction model for astaxanthin biosynthesis from glucose by P. equation 5).
rhodozyma:

12Glucose 1 4O2 1 14NADPH 1 48NAD1 5

C6H12O6 1 0.17NH3 1 3.26O2 5 2.65CH1.44O0.56N0.06 1
1Astaxanthin 1 32CO2 1 14NADP1 1
4.35H2O 1 3.35CO2
48NADH 1 12H2O 1 34H1 (4) The results are summarized in Table 6.
Cell assimilation equations with cosubstrates such as ATP
Equation 4 indicates that a large amount of NADH is pro-
and NADPH were then established by using the cell formula.
duced as a side product of the astaxanthin production, indicat-
For this, we made two assumptions: (i) YATP is 10.5 g per mol,
ing that astaxanthin production might also be repressed with
and (ii) a reductant for the synthesis of cell materials is
an increase in NADH accumulation due to the oxygen-limiting
NADPH. The basic equation can be expressed as follows:
conditions. This agrees with equation 2. On the other hand,
when a sufficient amount of oxygen is supplied to the cell, the C6H12O6 1 6zNH3 1 eNADPH 1 eH1 1 fATP 5
accumulated NADH can be reoxidized to NAD1, using 1 mol
of oxygen for 2 mol of NADH. In addition, since an oxygen 6CHxOyNz 1 eNADP1 1 fADP 1 gH2O
molecule is introduced into the astaxanthin molecule (6), ox-
ygen is also material requirement for astaxanthin biosynthesis. where e, f, and g are the stoichiometric constants. These con-
Therefore, it is considered that a sufficient supply of oxygen stants can be calculated based on the formula estimated above
can enhance astaxanthin production by preventing the accu- and the YATP concept. For example, the cell assimilation equa-
mulation of NADH and by supplying oxygen molecules as a tion for the batch culture with 80 g of glucose per liter can be
starting material, which agrees with equation 3. We therefore given as follows:
considered that this was why the astaxanthin production was C6H12O6 1 0.36NH3 1 0.42NADPH 1 0.42H1 1
enhanced with increasing oxygen uptake (respiration) and re-
pressed with increasing ethanol production (fermentation). 13.98ATP 5 6CH1.44O0.56N0.06 1
Second, we discuss the reason why the astaxanthin produc-
tion is enhanced by a high glucose supply at a fixed nitrogen 0.42NADP1 1 13.98ADP 1 2.76H2O (5)
concentration, that is, a high initial C/N ratio in the medium
with high levels of oxygen. An increase in astaxanthin produc- These results are also summarized in Table 6.
tion might be accompanied by variations in anabolism, such as As shown in Table 6, it was estimated that the nitrogen
YX/S and DX/DN, as shown in Table 4. To answer this question, content per gram (dry weight) of cells decreased with an in-
therefore, the differences in anabolism between P. rhodozyma crease in the glucose supply. The decrease in nitrogen content
cells with a low astaxanthin content and those with a high might be due to a decrease in the protein content of the cell.
astaxanthin content were analyzed by a stoichiometric proce- Actually, the protein content decreased with increasing glucose
dure. First, in order to estimate the difference in the growth supply. For example, the protein content was 0.62 6 0.02 g of
characteristics, the growth stoichiometric equations were es- protein per g (dry weight) of cells at 10 g of glucose per liter,
tablished. To establish these equations, we assumed that (i) but it decreased to 0.53 6 0.02 g per g at 80 g of glucose per
glucose was the sole carbon source and was converted primar- liter. As shown in Table 6, it was also estimated that the
ily to cell materials and carbon dioxide, (ii) ammonium was the NADPH consumption for anabolism per gram (dry weight) of
sole nitrogen source, and (iii) 5% of the cell mass is composed cell decreased with an excess glucose supply. Albers et al. (1)
of ash. The basic stoichiometric equation can be given as fol- and Bruinenberg et al. (7) have reported that when yeast is
lows: grown in a minimal medium containing glucose as a carbon
source and ammonium as a nitrogen source, a large amount of
C6H12O6 1 aNH3 1 bO2 5 6YCCHxOyNz 1 cH2O 1 dCO2 NADPH is consumed in amino acid synthesis. Therefore, it
was considered that a decrease in NADPH consumption might
where Yc is the ratio for carbon conversion from glucose to be due to a decrease in protein synthesis. The astaxanthin
cells (dimensionless) and a, b, c, and d are the stoichiometric synthesis requires a large amount of NADPH, as shown in
constants. Based on the calculated values of YX/S, DX/DN, equation 4. The decrease in NADPH consumption for primary
DX/DO2, and DX/DCO2 (Table 4), a, b, and d can be calculated. metabolism such as anabolism leads to an increase in the
YC can be rewritten by using YX/S, x, y, and z. Therefore, one amount of NADPH which can be utilized for astaxanthin pro-
can estimate c, x, y, and z by taking into account the balances duction. We therefore believe that this is why astaxanthin pro-
of C, O, H, and N. For example, the growth stoichiometric duction was enhanced by the high initial C/N ratio at high
equation for the batch culture with 80 g of glucose per liter can levels of oxygen.
be estimated as follows: As mentioned above, astaxanthin production might be in-
4478 YAMANE ET AL. APPL. ENVIRON. MICROBIOL.

creased by the repression of primary metabolism such as pro- Phaffia rhodozyma. J. Gen. Microbiol. 115:173–183.
tein synthesis, whereas cell growth might be decreased by the 15. Johnson, E. A., T. G. Villa, and M. J. Lewis. 1980. Phaffia rhodozyma as an
astaxanthin source in salmonid diets. Aquaculture 20:123–134.
repression of primary metabolism. Therefore, a two-stage fed- 16. Johnson, E. A., and W. A. Schroeder. 1996. Microbiol carotenoids, p. 119–
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separated from the cell growth stage, should be effective. This ogy. Springer-Verlag, Berlin, Germany.
hypothesis was supported by the results of the two-stage fed- 17. Kakizono, T., M. Kobayashi, and S. Nagai. 1992. Effect of carbon/nitrogen
ratio on encystment accompanied with astaxanthin formation in a green alga,
batch culture in the present study. Haematococcus pluvialis. J. Ferment. Bioeng. 74:403–405.
In conclusion, it appears that astaxanthin production in 18. Kobayashi, M., T. Kakizono, and S. Nagai. 1993. Enhanced carotenoid
P. rhodozyma is very much affected by cell metabolism, i.e., biosynthesis by oxidative stress in acetate-induced cyst cells of a green uni-
anabolism, respiration, and fermentation. This indicates that in cellular alga, Haematococcus pluvialis. Appl. Environ. Microbiol. 59:867–873.
19. Larsson, C., U. von Stockar, I. Marison, and L. Gustafsson. 1993. Growth
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to optimize the whole metabolic pathway in the cell, especially 175:4809–4816.
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