J. Trop. Agric. and Fd. Sc. 35(2)(2007): 287–A.M.
296 Musaalbakri, A. Ariff, M. Rosfarizan and A.K.M. Ismail
Kinetics of red pigment fermentation in 2-litre stirred
tank fermenter using different types and concentrations of
carbon sources
(Kinetik fermentasi pewarna merah di dalam fermenter berpengaduk 2 liter
menggunakan jenis dan kepekatan sumber karbon yang berbeza)
A.M. Musaalbakri*, A. Ariff**, M. Rosfarizan** and A.K.M. Ismail***
Key words: Monascus purpureus, kinetics, glucose, fructose, maltose, sucrose, red pigment,
non-growth associated
Abstract
Kinetics of red pigment fermentation by Monascus purpureus FTC 5391 using
various sources of carbon (glucose, fructose, maltose and sucrose) were analysed
using a model based on Logistic and Leudeking-Piret equations. Using the
optimal concentration of fructose, batch fermentation without pH control was
capable to produce slightly higher red pigment (20.70 UA500) as compared to
fermentation using glucose (20.63 UA500). In terms of overall productivity,
fermentation using fructose (0.153 UA500/h) was comparable to glucose
(0.122 UA500/h). The production of red-pigment by M. purpureus FTC 5391
appeared to be a non-growth associated process; whereby rapid red-pigment
production occurred during non-growth phase after the depletion of carbon in the
medium and the on-set of ethanol accumulation. It seemed that the red-pigment
was formed from the metabolism of ethanol accumulated in the culture.
Introduction
The composition ratios and productivity
of pigment can be influenced by the
Monascus strains, medium composition
and fermentation conditions in which they
are produced. Therefore, many attempts
have been made to enhance the production
of Monascus pigments through strain
improvement, different carbon source and
improvement of culture condition. The
Monascus pigments are a complex mixture
of at least six compounds. The quantity of
pigments produced by Monascus species is
greatly influenced by the types of carbon
and nitrogen sources used in medium
formulation; and also greatly influenced
by the culture condition such as the pH of
culture medium (Lee et al. 1994).
Although the cultural conditions for
the improvement of microbial pigment
production by Monascus sp., in general,
have been studied extensively, information
on the kinetics of production and
development of models to describe the
process in more meaningful ways remained
scarce. Monascus purpureus FTC 5391
*Biotechnology Research Centre, MARDI Headquaters, Serdang, P.O. Box 12301, 50774 Kuala Lumpur, Malaysia
**Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra
Malaysia, 43400 UPM Serdang, Selangor, Malaysia
***Department of Biotechnology Engineering, Faculty of Engineering, Universiti Islam Antarabangsa Malaysia,
Jalan Gombak, 53100 Kuala Lumpur, Malaysia
Authors’ full names: Musaalbakri Abdul Manan, Arbakariya Ariff, Rosfarizan Mohamad and
Mohamed Ismail Abdul Karim
E-mail: [email protected]
©Malaysian Agricultural Research and Development Institute 2007
287
Kinetics of red pigment fermentation
is a newly isolated strain and presently,
no attempt has been made to study its
growth kinetics. Furthermore, the culture
characteristic of this fungal strain could be
different from other fungus.
The objective of this study was to
develop simple models that can be used
to describe the kinetics of the growth
of M. purpureus FTC 5391 and its red
pigment production. The experimental data
from batch fermentations of M. purpureus
FTC 5391 using 2-litre stirred tank
fermenter was analysed in order to form
the basis for a kinetic model of the process.
The model may allow better understanding
of the fermentation process, so that suitable
fermentation technique and process control
strategy can be developed for improvement
of pigment production by M. purpureus
FTC 5391.
Materials and methods
Microorganism and medium
The monospore isolate MP 3 of
M. purpureus FTC 5391 (Musaalbakri 2004),
was used throughout this study. The fungus
was maintained on potato dextrose agar
(PDA) plate for 7 days at 37 °C. The
inoculum medium (YMP broth) consisted
of yeast extract (3 g/litre), malt extract
(3 g/litre), peptone (5 g/litre) and glucose
(20 g/litre). Four pieces of 4 mm mycelial
block of M. purpureus FTC 5391 were
used to inoculate the inoculum cultures in
250-ml flask containing 100 ml YMP broth.
The flasks were incubated in orbital shaker
at 37 °C, agitated at 250 rpm for 4 days.
The fermentation medium consisted of
monosodium glutamate (MSG), 12 g/litre;
K2HPO4, 2.5 g/litre; KH2PO4, 2.5 g/litre;
MgSO4.7H2O, 1.0 g/litre; KCl, 0.5 g/litre;
ZnSO4.7H2O, 0.01 g/litre; FeSO4.7H2O,
0.01 g/litre and MnSO4.H2O, 0.03 g/litre,
though the type and concentration of carbon
source added was varied according to the
requirement of each experiment. The sugar
solution was prepared and autoclaved
separately from basal medium and added
prior to the start of the fermentation
288
process. It is often best to sterilize sugar
solution separately because it may react
with ammonium ion and amino acid to
form black nitrogen containing compound
which will partially inhibit the growth
of many microorganisms (Stanbury and
Whitaker 1984).
Fermentation
The 2-litre stirred tank fermenter (Biostat
B. Braun, Germany) with a working volume
of 1.5 litres was used in this study. A single
six-bladed turbine impeller with a diameter
(D) of 52 mm mounted on the agitator shaft
was used for agitation. The fermenter was
equipped with temperature and dissolved
oxygen controllers. During the fermentation,
agitation speed (N) was fixed at 600 rpm
(impeller tip speed = ηND = 1.64 m/s).
In all experiments, the dissolved oxygen
tension (DOT) control level was set at 90%
(v/v) saturation by varying air flow rate
ranging from 0.1 litre/min (0.067 vvm) to
1.5 litres/min (1.0 vvm) using mass flow
controller. Dissolved oxygen tension (DOT)
was recorded continuously during the
fermentation. The initial pH of the culture
was adjusted to 6.5 and the culture pH was
not controlled but recorded continuously
during the fermentation. In all fermentations,
the temperature within the fermenter was
controlled at 37 °C. During fermentation,
samples were withdrawn at various time
intervals for analysis.
Analytical methods
Determination of cell concentration Cell
concentration was determined using filtration
and oven dry method. A known volume of
culture sample (5–10 ml) withdrawn from
the fermenter was filtered through a preweighed filter paper (Whatman No. 1) by
using vacuum pump. After drying period
of more than 24 h in an oven at 80 °C
i.e., until a constant weight was achieved,
the filter paper and cells were re-weighed
and the cell dry weight was calculated by
difference.
A.M. Musaalbakri, A. Ariff, M. Rosfarizan and A.K.M. Ismail
Glucose and other reducing sugar
analysis Glucose concentration in the
culture broth for fermentation using glucose
as the sole carbon source was measured
by using Glucose Analyzer (YSI 2700
Select Biochemistry Analyzer). Samples
were prepared by filtering the supernatant
through sep – pack to remove the particles
and pigments that may interfere with the
determination. For fermentation using
fructose, maltose and sucrose as carbon
source, each reducing sugar concentration in
the culture broth was determined using high
performance liquid chromatography (HPLC)
(Waters 2690, USA) with refractive index
detector (Waters 2410). Separation of sugars
was obtained using NH2 Column (MERCK)
and 80% acetonitrile in deionised water as
a stationary and mobile phase, respectively.
The temperature of the column was
maintained at ambient temperature and the
flow rate was fixed at 1.0 ml/min. Samples
were prepared by filtering fermentation
broth with Whatman nylon filter paper
(0.2 µm pore size; 47 mm diameter). The
concentration of reducing sugars (fructose,
maltose and sucrose) was quantified using a
pre-determined calibration curve.
Determination of red pigment Samples
collected during the fermentation were
centrifuged at 3,000 rpm for 10 min using
laboratory centrifuge (Centrifuge 5810R,
Germany). The red pigment was present
in both fractions, filtrate and cell pellet. In
order to measure red pigment in cell pellet,
extraction of the pigment was carried out
using 95% (v/v) ethanol. The method of
extraction was used as follows: 10 ml of
ethanol was added to 1 g wet cell in
20-ml test tube, shaken for a while and
then kept at room temperature overnight.
The mixture was then filtered through
a filter paper (Whatman No. 1) and the
filtrate was used for pigment determination.
For the measurement of absorbance for
filtrate from culture broth, uninoculated
medium was used as a blank while for
filtrate from the extract, ethanol was used
as blank. The wavelength at 500 nm
represents absorption maxima for the red
pigment. Whenever necessary, the samples
were diluted with distilled water (filtrate)
or ethanol (extract) prior to absorbance
measurement. The pigment production was
calculated by multiplying the absorbance
units by the dilution factor. The spectra
of the red pigment were measured using
a Cecil CE 2502 2000 series scanning
spectrophotometer.
Determination of ethanol Ethanol was
measured using high performance liquid
chromatography (HPLC) (Waters 2690,
USA) with refractive index detector (Waters
2410). The separation of ethanol was
obtained by using Biorad aminex HPX-87H
cation-exchange resin column (300 x 7.8 I.D.)
as stationary phase. The mobile phase was
7 mM sulphuric acid (H2SO4). Flow rate of
the mobile phase and column temperature
was controlled at 1.0 ml/min and 50 °C
respectively. Samples were prepared by
filtering fermentation broth with Whatman
nylon filter paper (0.2 µm pore size, 47 mm
diameter). The concentration of ethanol was
quantified using a pre-determined calibration
curve.
Mathematical method
The following simplified batch fermentation
kinetic models for cell growth, substrate
consumption and product formation
based on Logistic and Luedeking-Piret
equations, which have been described
elsewhere (Weiss and Ollis 1980), were
used to evaluate the kinetics of red pigment
production by M. purpureus FTC 5391.
The fungus, M. purpureus FTC 5391,
employed in pigment fermentation was
very stable as shown by high viability,
even at extreme growth conditions. From
preliminary kinetics study, it was found that
growth of M. purpureus FTC 5391 followed
logistic equation. Thus, the simplified
batch fermentation kinetic models for cell
growth, substrate consumption and product
formation based on logistic and Leudeking289
Kinetics of red pigment fermentation
Piret equation, are proposed for red pigment
fermentation by M. purpureus FTC 5391
and they expressed as follows:
Cell growth
dX/dt = [µmax(1-X/Xmax)]X (1)
Substrate consumption -dS/dt = α(dX/dt) + βX
(2)
Product formation
dP/dt = m(dX/dt) + nX
(3)
The batch fermenter models (equations 1-3)
were fitted to the experimental data by nonlinear regression with MS Excel computer
software. The model parameter values were
evaluated by solving equation (1–3). The
predicted values were then used to simulate
the profiles of cell, substrate and product
concentrations during the fermentation. To
determine whether the deviations between
the experimental and calculated data are
significant or not-significant, statistical
analysis (unpaired T-test) was also carried
out by using SAS program (SAS Inst. 1990).
Results and discussion
Effect of different carbon sources
A typical time courses of M. purpureus
FTC 5391 fermentation in 2-litre stirred
tank fermenter using different types of
sugar (glucose, fructose, maltose and
sucrose) are shown in Figure 1, which also
includes the simulation curves calculated
according to the proposed kinetic models
(Eqs. 1–3). The models fitted fairly well
with more than 94% confidence to the
experimental data. Comparison of the
performance and the kinetic parameter
values of red pigment fermentation using
different types of sugar in 2-litre stirred
tank fermenter are shown in Table 1. The
kinetic parameter values obtained can be
used to verify the experimental data of
red pigment production by M. purpureus
FTC 5391 using various sugars. All the
carbon sources tested except sucrose were
able to support growth of M. purpureus
FTC 5391 and red pigment production.
Growth and red pigment production was
greatly reduced in fermentation using
sucrose, suggesting that sucrose was a poor
carbon source for M. purpureus FTC 5391.
290
The unability of the fungus to hydrolyse
sucrose to glucose and fructose may be
one of the possible reasons for reduced
fermentation performance when sucrose
was used as carbon source. When glucose,
fructose and maltose were used as carbon
sources, the maximum concentration of
cell obtained were 13.2, 14.24 and 11.7
g/litre, respectively. However, the maximum
specific growth rate (μmax) with an average
value of 0.062/h, was not significantly
different with the different sugars used.
Red pigment production obtained in
fermentation using glucose (20.63 UA500)
and fructose (20.70 UA500), was not
significantly different, which gave an overall
pigment productivity (P) of 0.122 and
0.153 UA500 red pigment/h, respectively.
Monascus purpureus FTC 5391 was
capable to utilize maltose for growth and
red pigment production, indicating that the
fungus secreted sufficient glucoamylase
enzyme for hydrolysis of maltose to glucose
in the prevailing fermentation conditions.
It is important to note that excellent growth
(14.24 g/litre) and red pigment production
(20.70 UA500) was obtained when fructose
was used as a carbon source. Chen and
Johns (1994) reported that the medium with
fructose as carbon source proved to be the
most suitable for cell growth and pigment
production by M. purpureus, followed with
maltose and glucose. They also reported
that growth and pigment production were
inhibited when sucrose and lactose were
used as carbon sources.
The rate of carbon source consumption
for fermentation using different carbon
sources are shown in Figure 1c. As can be
seen, M. purpureus FTC 5391 was able
to utilize various types of carbon sources
for growth and red pigment production
except sucrose. The highest consumption
rate was observed in medium using
glucose as a carbon source followed by
fructose and maltose. All glucose supplied
in the culture (100%) was consumed for
cell growth after 72 h of cultivation. The
ability of M. purpureus FTC 5391 to utilize
A.M. Musaalbakri, A. Ariff, M. Rosfarizan and A.K.M. Ismail
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25 b)
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Figure 1. Batch red pigment fermentation by M. purpureus FTC 5391 in 2-litre stirred tank fermenter
using different carbon sources, which also includes the comparison between the calculated and
experimental data. a) Cell concentration, b) Red pigment production, c) Substrate consumption, d) pH
profile, e) Profile of dissolve oxygen tension and f) Ethanol production, (solid lines represent data
according to equations 1–3)
disaccarides indicated that this strain
also produced enzymes which degraded
disaccarides into monosaccarides for further
use in red pigment production. About 97%
of disaccarides (maltose) was consumed
for both cell growth and red pigment
production. Although maltose was consumed
at the same rate as other carbon sources, the
production of red pigment was low. This
might be due to the increment of culture pH
to above 5, which became unfavourable for
red pigment production (Figure 1d).
Result from this study indicates that
locally isolated M. purpureus FTC 5391
was able to utilize various types of carbon
sources except sucrose for red pigment
production. However, glucose was found to
be the best carbon source for red pigment
production in which the maximum cell
concentration (13.2 g/litre) and maximum
291
Kinetics of red pigment fermentation
Table 1. Comparison of the performance and kinetic parameter values of red
pigment fermentation in 2-litre stirred tank fermenter using different types of
carbon sources
Kinetic parameter values
Types of carbon sources
Glucose
Maltose
Fructose
Xmax (g/litre)
13.2 11.7
14.24
X0 (g/litre) 0.25 0.25 0.25
μmax (h-1) 0.065 0.060 0.060
P0 (UA500) 0.085 0.085 0.085
Pmax (UA500)
20.63 9.43
20.70
Yx/s (g cell/g carbon) 0.248 0.227 0.383
P (g cell/litre.h) 0.077 0.101 0.102
Yp/s (UA500/g.litre) 0.411 0.181 0.575
P (UA500/h) 0.122 0.068 0.153
α (g carbon/g cell) 4.0 4.0 2.2
β (g carbon/g cell.h) 0.005 0.005 0.005
m (UA500 red pigment/g cell) 0 0 0
n (UA500 red pigment/g cell.h) 0.02 0.012 0.04
red pigment concentration (20.63 UA500) was
obtained after 7 days of fermentation. As
presented and discussed earlier, red pigment
production by M. purpureus FTC 5391 using
glucose in the stirred tank fermenter was
non-growth associated system (Musaalbakri
et al. 2005b). In this study, it was found
that pigment fermentation using maltose,
fructose and sucrose was also a non-growth
associated system, as indicated by zero value
of growth associated rate constant for red
pigment formation (m) for all fermentations.
The non-growth associated rate constant for
red pigment formation (n) for fermentation
using fructose was comparable to glucose,
but the value was about two times higher for
fermentation using fructose.
Effect of glucose concentration
Figure 2 shows the profile of growth, red
pigment production, glucose consumption,
pH and DOT and ethanol production during
fermentation of M. purpureus FTC 5391
using different glucose concentrations. There
was significant difference on growth and
red pigment production for fermentation
using 50–150 g/litre glucose. The highest
maximum cell concentration, Xmax,
(20.48 g/litre) was obtained in fermentation
using 75 g/litre glucose. In terms of cell
292
Sucrose
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0.549
0.059
0.188
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2
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0.0045
yield, fermentation using 75 g/litre glucose
gave the highest value (0.27 g cell/g
glucose) than for fermentation using
50 g/litre glucose (0.248 g cell/g glucose).
On the other hand, red pigment production
was decreased almost linearly with
increasing glucose concentration. At a range
of glucose concentration studied, the highest
red pigment production (20.63 UA500) was
obtained in fermentation using 50 g/litre
glucose which gave an overall productivity
of 0.122 UA500 red pigment/h. These values
were slightly higher than those obtained in
fermentation using 100 g/litre and 75 g/litre
glucose.
In terms of pigment yield, fermentation
using 75 g/litre glucose gave lower value
(0.343 UA500 red pigment/g glucose) than
for fermentation using 50 g/litre glucose
(0.411 UA500 red pigment/g glucose).
Glucose was exhausted after 132 h at the
time where red pigment concentration
became maximum in fermentation using
50, 75 and 100 g/litre glucose. Although
growth was not inhibited in fermentation
using 120 g/litre and 150 g/litre glucose,
production of red pigment was reduced,
even though all glucose was consumed by
M. purpureus FTC 5391. Furthermore, the
pH of the cultures using different glucose
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▲
■
▲
■
●
6
5
4
3
2
1
0
0
●
120
100
▲
●
■
●
◆
c)
●
20
0
▲
●
■
◆
●
50
60
40
●
●
◆
■
▲
●
◆
■
●
▲
●
■
●
◆
▲
▲
◆
◆
▲
◆
◆
◆
pH
Glucose (g/litre)
140
◆
●
●
▲
■
●
●
◆
▲
160
pO2 (5)
▲
▲
▲
Red pigment (UA500)
20
0
20 b)
18
16
14
12
10
8
6
4
2
●
◆ ◆
0 ▲■●● ▲●■
0
a)
Ethanol (g/litre)
Cell concentration (g/litre)
25
■
■
●
●
■
0
T (h)
●
■
■
●
▲
◆
●
▲
◆
●
▲
●
■
▲
◆
50
▲
◆
●
●
■
◆
■
100
■
◆
■
◆
■
◆
●
◆
■
150
●
◆
■
200
Figure 2. Batch red pigment fermentation by M. purpureus FTC 5391 in 2-litre stirred tank fermenter
using different concentrations of glucose, which also includes the comparison between the calculated
and experimental data. a) Cell concentration, b) Red pigment production, c) Glucose consumption,
d) pH profile, e) Profile of dissolve oxygen tension and f) Ethanol production, (solid lines represent data
according to equations 1–3)
concentrations throughout the fermentation
was ranged from 6.5 to 9. This range
of pH was reported as a suitable pH as
discussed by Musaalbakri et al. (2005a).
It is interesting to note that the fermentation
time for fermentation in 2-litre stirred tank
fermenter was the same with the time taken
for fermentation in shake flask (Musaalbakri
et al. 2005b).
Figure 2(a–c) also shows the
comparison of calculated and experimental
data for fermentation by M. purpureus
FTC 5391 in a 2-litre stirred tank fermenter
using different concentrations of glucose.
The performance and kinetic parameter
values of all fermentations are presented
in Table 2. In all cases, the models fitted
well to the experimental data of growth,
glucose consumption, and red pigment
production. The maximum specific growth
rate (μmax) was not significantly different
for the different concentrations of glucose
used with an average value of 0.06/h. The
value of growth-associated rate constant
293
Kinetics of red pigment fermentation
for glucose consumption (α) was increased
with increasing glucose concentration up
to 150 g/litre. On the other hand, the value
of non-growth associated rate constant for
glucose consumption (β) was the same. The
value of non-growth associated rate constant
for red pigment production (n) in 50 g/litre
glucose (0.02 UA500 red pigment/g cell) was
about two times higher than the value from
other concentrations.
Relationship between red pigment
production and ethanol accumulation
Figure 1f and Figure 2f show the profile
of ethanol production during M. purpureus
FTC 5391 fermentation using different
types of carbon sources and different
concentrations of glucose respectively. In all
cases, it can be seen that rapid consumption
of carbon, and the concomitant rapid
reduction in DOT occurred during active
growth. The on-set of ethanol production
was observed when either glucose in the
culture became depleted and/or DOT
level dropped to very low level i.e. almost
zero. Red pigment production occurred
during growth on carbon source supplied
in the medium and also during growth on
ethanol accumulated in the culture (i.e.
after exhausting carbon source supplied).
However, higher red pigment production
with reduced growth rate, in some cases
growth ceased, occurred during utilization of
ethanol as carbon source.
The ability of Monascus species to
produce ethanol is well known, having
been extensively used in Asia for alcoholic
beverage manufacture (Lin and Iizuka
1982). Ethanol production was enhanced
when mass transfer limits the oxygen
consumption and when anoxic conditions
occurred in the cultures (dissolved oxygen
concentration close to zero) (Figure 1e and
Figure 2e). In the presence of high glucose
concentrations, as for certain Monascus
species, ethanol is also produced which
inhibits pigment production (Pastrana et
al. 1995). Chen and Johns (1994) also
reported that ethanol production led to a
corresponding decrease in cell yield.
Conclusion
Unstructured models based on Logistic and
Leudeking-Piret equations were proposed to
describe growth, substrate consumption and
red pigment production by M. purpureus
FTC 5391. The proposed models based on
Logistic and Leudeking-Piret equations were
found suitable to describe M. purpureus
FTC 5391 fermentation for production of
red pigment using various types of carbon
sources and various concentrations of
Table 2. Comparison of the performance and kinetic parameter values of red pigment
fermentation in 2-litre stirred tank fermenter using different concentrations of glucose
Kinetic parameter values
Glucose concentrations (g/litre)
50
75
100
120
150
Xmax (g/litre)
X0 (g/litre)
μmax (h-1)
P0 (UA500)
Pmax (UA500)
Yx/s (g cell/g glucose)
P (g cell/litre.h)
Yp/s (UA500/g.litre)
P (UA500/h)
α (g glucose/g cell)
β (g glucose/g cell.h)
m (UA500 red pigment/g cell)
n (UA500 red pigment/g cell.h)
13.2
0.25
0.065
0.085
20.63
0.248
0.077
0.411
0.122
4.0
0.005
0
0.02
20.48
0.25
0.06
0.085
7.25
0.270
0.12
0.343
0.102
5.0
0.005
0
0.01
15.2
0.25
0.05
0.085
15.47
0.150
0.096
0.154
0.107
7.0
0.005
0
0.01
14.85
0.25
0.055
0.085
10.66
0.122
0.122
0.088
0.098
9.0
0.005
0
0.01
13.3
0.25
0.055
0.085
12.62
0.087
0.091
0.084
0.087
13.0
0.005
0
0.01
294
A.M. Musaalbakri, A. Ariff, M. Rosfarizan and A.K.M. Ismail
glucose. The models can be used to generate
parameter values that may be used to verify
the experimental data; and also simulation
and optimization of the process.
Locally isolated M. purpureus
FTC 5391 was able to utilize various types
of carbon sources except sucrose for red
pigment production. Using the optimal
concentration of fructose, batch fermentation
without pH control was capable to produce
slightly higher red pigment (20.70 UA500)
as compared to fermentation using
glucose (20.63 UA500). In terms of overall
productivity, fermentation using fructose
(0.153 UA500/h) was comparable to glucose
(0.122 UA500/h).
Acknowledgement
This study was funded by IRPA (Research
Grant No. 1900301634).
References
Chen, M.-H. and Johns, M.R. (1994). Effect
of carbon source on ethanol and pigment
production by Monascus purpureus. Enzyme
Microbiology Technology 16: 584–90
Lee, Y.-K., Lim, B.-L., Ng, Ai-L. and Chen, D.-C.
(1994). Production of polyketide pigments by
submerged culture of Monascus: Effects of
substrates limitations. Asia Pacific Journal of
Molecular Biology and Biotechnology 2(1):
21–6
Lin, C.-F. and Iizuka. H. (1982). Production
of extracellular pigment by a mutant of
Monascus kaoliang sp. novel. Applied and
Environmental Microbiology 43(3): 671– 6
Musaalbakri, A.M. (2004). Kinetics of red-pigment
fermentation by Monascus purpureus FTC
5391 in shake flask culture and stirred tank
fermenter using different carbon and nitrogen
sources. MS Thesis, Institute of Biosciences,
Universiti Putra Malaysia, Serdang
Musaalbakri, A.M., Arbakariya, A., Rosfarizan, M.
and Ismail, A.K. M., (2005a). Fermentation
conditions affecting growth of Monascus
purpureus FTC 5391. J. Trop. Agric. and Fd.
Sc. 33(2): 261–76
Musaalbakri, A.M., Arbakariya, A., Rosfarizan, M.
and Ismail, A.K.M. (2005b). Kinetics and
modeling of red pigment fermentation in
2-litre stirred tank fermenter using glucose as
a carbon source. J. Trop. Agric. and Fd. Sc.
33(2): 277–84
Pastrana, L., Blanc, P.J., Santerre, A.L., Loret,
M.O. and Goma, G. (1995). Production of
red pigments by Monascus ruber in synthetic
media with a strictly controlled nitrogen
source. Process Biochemistry 30(4): 333–41
SAS Inst. (1990). SAS user’s guide: Statistics
version 6. Cary, NC: SAS Institute Inc.
Stanbury, P.F. and Whitaker, A. (1984). Principles
of fermentation technology. New York:
Pergamon Press
Weiss, R.M. and Ollis, D.F. (1980). Extracellular
microbial polysaccharides I. Substrate,
biomass and product kinetic equations
for batch xantham gum fermentation.
Biotechnology and Bioengineering 22:
859– 64
295
Kinetics of red pigment fermentation
Abstrak
Kinetik fermentasi pewarna merah oleh Monascus purpureus FTC 5391
menggunakan pelbagai sumber karbon (glukosa, fruktosa, maltosa dan sukrosa)
dianalisis menggunakan model berdasarkan persamaan Logistic dan LeudekingPiret. Dengan menggunakan kepekatan fruktosa yang optimum, fermentasi
sesekelompok tanpa kawalan pH menghasilkan hanya sedikit peningkatan
pewarna merah (20.70 UA500) dibandingkan dengan fermentasi menggunakan
glukosa (20.63 UA500). Dari segi produktiviti keseluruhan, fermentasi
menggunakan fruktosa (0.153 UA500/j) adalah setanding dengan glukosa (0.122
UA500/j). Penghasilan pewarna merah oleh M. purpureus FTC 5391 ditunjukkan
sebagai proses pertumbuhan tidak berkait, iaitu penghasilan pewarna merah
lebih pantas semasa fasa tiada pertumbuhan iaitu selepas karbon di dalam kultur
semakin berkurangan dan etanol bertambah. Ia seolah-olah pewarna merah
terbentuk daripada kesan metabolisme terhadap etanol di dalam kultur.
Abbreviation
α Growth-associated rate constant for glucose consumption (g glucose/g cell)
β Non-growth-associated rate constant for glucose consumption (g glucose/g cell.h)
µmax Maximum or initial specific growth rate (h-1)
m Growth associated rate constant for red pigment production (UA500/g cell)
n Non-growth-associated constant for red pigment production (UA500/g cell.h)
P Productivity
P0 Initial red pigment concentration (UA500)
Pmax Maximum red pigment concentration (UA500)
S0 Initial substrate concentration (g/litre)
Smax Substrate concentration (g/litre)
X0 Initial cell concentration (g/litre)
Xmax Maximum cell concentration (g/litre)
Yp/s Yield of red pigment based on glucose consumed (UA500/g.litre)
Accepted for publication on 13 December 2006
296