Bioprocess Engineering 17 (1997) 7579 Springer-Verlag 1997

A new process for red pigment production by submerged culture

of Monascus purpureus
M. Hamdi, P.J. Blanc, M.O. Loret, G. Goma

75
Abstract Formation of red pigment by Monascus purpureus via diauxic growth on glucose and ethanol in
submerged culture was optimized based on inoculum
preparation and culture medium. A vegetative inoculum
was prepared from spores grown on ethanol. The optimized culture medium was low in phosphates, and had an
initial pH of 5.5. The characteristics of Monascus purpureus grown on glucose and on ethanol were compared: the
specific consumption rate of glucose (qG) was higher than
the specific consumption rate of ethanol (qE), whereas the
specific growth rate was greatest with ethanol. The specific
production rate of red pigment (pOD) and pigment yield
(YOD/s) with glucose was twice that with ethanol. A novel
fermentation process was developed with M. purpureus
initially grown with controlled ethanol formation, and
consumption of the latter during pigment formation.
List of symbols

DO
qG
qE
l
pE
pOD
Yx/s
YOD/s

(%)
Dissolved oxygen
(g/g.h)
Specific consumption of glucose
(g/g.h)
Specific consumption of ethanol
(h1)
Maximum specific growth rate
(g/g.h)
Specific production rate of ethanol
(ODU/g.h) Maximum specific production rate of
red pigment
(g/g)
Yield of biomass on substrate
(ODU/g) Yield of red pigment on ethanol

1
Introduction
Study of the natural red food colorants has intensified and
the objective being to replace certain synthetic ones.
Monascus sp. red pigment has been recently utilized in the
orient for making red rice wine, red soybean cheese, and
red chinese rice [1]. The production of Monascus pigment
as food coloring agent by both submerged and agar surface

cultures have been studied extensively in the past 10 years

[1, 2, 3, 4]. Yields in submerged culture are affected by
medium composition, pH and agitation [2, 3]. A chemically-defined medium supporting rapid and intensive red
pigment production, using glutamate as nitrogen source
was developed, and shifted the site of pigment from being
predominantly cell-bound to being mainly extra cellular
[4]. We focus on red pigment production by Monascus
purpureus (this work) and M. ruber [5] grown in a
submerged culture in both defined and complex media.

2
Material and methods
2.1
Microorganism and inoculum preparation
Monascus purpureus CBS 10907 was maintained on potato
dextrose agar (PDA) (Difco, Detroit, MI.). Spores were
prepared by growth on PDA in an Erlenmeyer flask for 6
days at 30 C. Spores were washed from the agar with
sterile water and glass beads. Cultures were inoculated
with a spore suspension (0.5 ml) in 250-ml Erlenmeyer
flasks (40/250 ml) of optimized medium (see below,
Table 1) and incubated on a rotary shaker (150 rpm) at
30 C.
2.2
Growth conditions
Batch cultures were performed in 2 l fermentors (S.G.I.,
Toulouse, France) containing 1.5 l optimized medium
(Table 1) at 30 C. The agitation system was a Rushton-like
turbine. The dissolved oxygen concentration was kept
constant or modified by changing the impeller speed or by
aeration flow rate which was measured with a gas mass
flow meter. The DO concentration was maintained between 60 and 80% of saturation level. The pH was not
maintained constant.

2.3
Analytical methods
Biomass concentration was determined by filtering the
P.J. Blanc, M.O. Loret, G. Goma
Departement de Genie Biochimique et Alimentaire.
mycelia through a glass fiber membrane Whatman GF/C,
UA CNRS N 544. INSA, Complexe Scientifique de Rangueil. washed with water, dried under low pressure at 60 C for
31077 Toulouse Cedex, France
24 h, and then weighed. The water soluble red pigment
were estimated by measuring the optical density at 480 nm
M. Hamdi
using a Kontron Uvikon spectrophotometer.
Ecole Superieure des Industries Alimentaires,
Ethanol concentration was determined using a Intersmat
58 Avenue Alain Savary. 1003 Tunis, Tunisia
IGC 120 DFL gas chromatograph equipped with a flame
Correspondence to: P.J. Blanc
ionization detector: column (2 m 3.2 mm) packed with a
Received: 23 September 1996

Bioprocess Engineering 17 (1997)

Table 1. Media used for inoculum preparation and red pigment formation

76

Nutriments or salts

Inoculum preparation

Red pigment formation

Glucose (g/l)
Ethanol (g/l)
Monosodium glutamate (g/l)
Potassium dihydrogenophosphate (g/l)
Dipotassium hydrogenophosphate (g/l)
Potassium chlorure (g/l)
Magnesium sulfate (g/l)
Iron sulfate (mg/l)
Zinc sulfate (mg/l)
Manganese sulfate (mg/l)
pH
Inoculum

25
4
2.5
2.5
0.5
1
10
10
3
5
Spores (105/ml)

2050
213*
12.4
0.125
0.125

10
10
3
5.5
Free mycelium 2%(V/V)

*Successive addition during red pigment formation: See Figs. 3 and 5

Poropack QS 80100 mesh, with nitrogen as the carrier

gas. Injector, detector, and column temperatures were 260,
260, and 195 C respectively. Quantitative data were obtained by peak integration with an Intersmat ICR 1B after
calibration with propanol (10 g/l) internal standard.
Glucose analyses of broth supernatants (centrifuged and
diluted) were by using a glucose analyzer (YSI Model 27,
SGI, France). Dissolved oxygen (DO) concentrations were
measured with an Ingold amperometric probe. Citric,
succinic, acetic and malic acids were analysed by HPLC:
Waters liquid chromatograph equipped with an ION organic acids column and a refractometer 410. Elution was
carried out with an aqueous solution of H2SO4 10 mM at a
flow rate of 0.4 ml/min.

3
Results and discussion
3.1
Optimization of media for inoculum preparation
and red pigment formation
Inocula can be crucial in fungus culture. Preliminary runs
based on the factorial design experiments showed that the
medium of Lin and Demain [4] is good for inoculum
preparation when the monosodium glutamate (MSG) was
reduced to 4 g/l, and source carbohydrates was changed to
ethanol, and the initial pH changed from 5.5 into 5. The
ethanol (23%) yields better vegetative growth than glucose. At higher concentration of ethanol, lesser biomass
was produced with formation of larger pellets (Fig. 1).
Pellets may originate from spores coalescing, freshly germinating spores aggregating, or by mycelial entanglement.
Optimum concentration of spores to inoculate the ethanol
medium is about 105 spores/ml whereas with glucose it is
higher 107 spores/ml. The ethanol is more suitable source
carbohydrate for inoculum preparation than glucose, because it gives twice mycelium concentration and it requires lesser spores for inoculation. The optimum initial
pH of 5 favors spore germination, yields a free mycelium,
higher biomass but lesser red pigment production (Fig. 1).
Indeed, Lin [1] showed that maximum cell yield was obtained at pH 5, while the maximum pigment yield was at 6.
At pH values above 5.5, cell walls of most microorganisms
are negatively charged, tending to cause separation of the
cells by electrostatic repulsion [6]. Under optimal conditions for inoculum preparation M. purpureus gave max-

Fig. 1. Influence of initial concentration of ethanol and initial pH

on the biomass ( ) and red pigment production (j) by
Monascus purpureus growth on ethanol

imum biomass in 4 days, a maximum specific growth rate

of around 0.083 h1, with biomass yield of 8.9 g/l. The
pH of the medium increased slowly from 5 up to 5.32 and
red pigment was not greater than 0.7 ODU/ml.
Optimization of red pigment production in Erlenmeyer
flasks using a vegetative inoculum showed 2% v/v as the
optimal inoculum (Table 1). Relatively low amounts of
potassium dihydrogenophosphate (50 mg/l) and dipotassium hydrogenophosphate (50 mg/l) were sufficient for
high red pigment production. An initial pH of 5.5 favored
mycelial growth, and as it rose it resulted in improved red
pigment formation. Indeed, in submerged culture, pigments normally remain in the mycelium due to low solu-

M. Hamdi et al.: Production of MonascusI red pigment

bility in usually acidic medium [7]. Yet, Mak et al. [8]

noted that high pH might facilitate the pigment removal
from mycelium and was associated with the chemical
conversion of orange to red. This optimized medium for
red pigment formation was used in further experiments.
In preliminary studies several carbon sources were
compared with regard to red pigment production (data
unreported). Glucose and ethanol appeared useful substrates. When the carbon source was a mixture of glucose
and ethanol (Fig. 2), growth was in two stages diauxie.
Glucose gave increase in biomass and ethanol, while
ethanol resulted in growth of biomass plus pigment formation (Fig. 2).

3.2
Red pigment production in fermentor
Both substrates glucose and ethanol were studied for red
pigment production in fermentor. Fermentors inoculated
with a vegetative mycelial inoculum reduced the lag phase,
and gave a higher specific growth rate than inocula of a
spore suspension or vegetative pellets (data unreported).
Culture parameters obtained with glucose are illustrated
in Figs. 3 and 4. The biomass concentration increased in
two stages corresponding to glucose consumption and
ethanol formation for 75 h, and then ethanol consumption
and red pigment formation. The specific consumption rate
of glucose (qG) increased continuously from 0.1 to 0.3 g/
g.h. The specific production rate of ethanol from glucose
(pE) ranged from 0.05 to 0.08 g/g.h, and could be increased
by decreasing the DO concentration [9]. The specific
growth rate on glucose decreases from 0.06 to 0.02 h1,
whereas on the accumulated ethanol the mean specific
growth rate was around 0.025 h1. After 80 h, the biomass
decreases probably because of pigment removal from
mycelium. The red pigment formation is initiated especially when glucose was completely exhausted and corresponding to the beginning of ethanol consumption

Fig. 2. Time courses of glucose (h), ethanol (m), biomass (), and
red pigment (j), during Monascus purpureus growth on mixed
ethanol and glucose in flask cultures

77

Fig. 3. Time courses of glucose (h), ethanol (m), biomass (),

red pigment (j) and pH () during Monascus purpureus growth
on glucose in fermentor

Fig. 4. Time courses of l (), qG (h), pE (), qE (m) and pOD (j)
during Monascus purpureus growth on glucose in fermentor

Bioprocess Engineering 17 (1997)

78

(Fig. 3). The specific consumption rate of ethanol (qE) was

0.050.08 g/g.h. The red pigment formation started after
50 h and reached 8.5 ODU/ml at the end of fermentation.
The specific production rate of red pigment (pOD) continuously increased and reached 0.1 ODU/g.h. Growth of
M. purpureus on glucose, showed that red pigment formation was not coupled to the glucose consumption but to
ethanol consumption. The pH increases continuously
during red pigment formation and reached 6.6 at the end
of culture. Red water-soluble Monascus pigment is unstable at acidic pH [10].
With ethanol as sole carbon source, ethanol consumption, biomass and red pigment formation progressed simultaneously (Figs. 5 and 6). The specific consumption
rates of ethanol (qE) were 0.2 g/g.h for the first 50 h but
only 0.1 g/g.h during red pigment production. The specific
growth rate on ethanol ranged from 0.01 to 0.06 h1 during
50 h, whereas the mean specific growth rate during red
pigment formation averaged 0.015 h1. The biomass decrease was lesser than with glucose and started after 100 h
of culture. The red pigment formation began after 50 h
and reached 7.8 ODU/ml at the end of fermentation. The Fig. 6. Time courses of l (), qE (m) and pOD (j) during Monascus
specific production rate of red pigment (pOD) continuously purpureus growth on ethanol in fermentor
increased and reached 0.05 ODU/g.h. In contrast to the
fermentations with glucose, the pH increased but gave a
decrease after each addition of ethanol. Higher citric,
malic, succinic and acetic acids concentrations were produced with ethanol fermentation than with glucose fermentation (Fig. 7).
The growth of M. purpureus on glucose and on ethanol
are summarized in Table 2. Glucose is more suitable for
red pigment production than ethanol, but ethanol can be

Fig. 7. Time courses of citric acid (j), malic acid (r), succinic acid
(h) and acetic acid (m) during Monascus purpureus growth on
glucose (above) and on ethanol (below) in fermentor

Fig. 5. Time courses of ethanol (m), biomass (), red pigment (j)
and pH () during Monascus purpureus growth on ethanol in
fermentor

supplemented after exhaustion of ethanol initially synthetized from glucose. The specific consumption rate of
glucose (qG) was higher than the specific consumption rate
of ethanol (qE) during the growth step. The biomass from
glucose was more favorable to red pigment formation than
that obtained from ethanol. In both fermentations, the
pigments were formed from the ethanol. The specific
production rate of red pigment (pOD) and the yields
(YOD/E) obtained with fermentation of glucose was twice of

M. Hamdi et al.: Production of MonascusI red pigment

Table 2. Growth characteristics of M. purpureus on glucose and on ethanol

Growth characteristics

Glucose

Ethanol

Specific consumption of glucose qG (g/g.h)

Specific consumption of ethanol qE (g/g.h)
Maximum specific growth rate l (h1)
Specific production rate of ethanol pE (g/g.h)
Maximum specific production rate of pigments pOD (ODU/g.h)
Yield of biomass on substrate Yx/s (g/g)
Yield of red pigment on ethanol YOD/s (ODU/g)

0.10.3
0.050.08
0.04
0.05 -0.08
0.1
0.15*
0.81.1

0.10.2
0.06

0.05
0.3
0.50.7

*depending of the partial oxygen pressure during fermentation

79

that obtained with ethanol fermentation, though the spe- References

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