Appl Microbiol Biotechnol (2007) 73:10541058

DOI 10.1007/s00253-006-0577-1

BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING

Optimization of nutrient parameters for lovastatin

production by Monascus purpureus MTCC 369 under
submerged fermentation using response surface methodology
Sadik Ali Sayyad & Bibhu Prasad Panda &
Saleem Javed & Mohd Ali

Received: 28 April 2006 / Revised: 6 July 2006 / Accepted: 10 July 2006 / Published online: 22 September 2006
# Springer-Verlag 2006

Abstract Lovastatin, an inhibitor of HMG-CoA reductase,

was produced by submerged fermentation using Monascus
purpureus MTCC 369. Five nutritional parameters screened
using PlackettBurman experimental design were optimized by
BoxBehnken factorial design of response surface methodology
for lovastatin production in shake flask cultures. Maximum
lovastatin production of 351 mg/l were predicted in medium
containing29.59g/ldextrose,3.86g/lNH4Cl, 1.73 g/l KH2PO4,
0.86 g/l MgSO47H2O, and 0.19 g/l MnSO4H2O using
response surface plots and point prediction tool of DESIGN
EXPERT 7.0 (Statease, USA) software.
Keywords Lovastatin . Monascus purpureus .
Optimization . Response surface methodology
Introduction
Lovastatin, a hypocholesterolemic agent, competitively
inhibits the rate-limiting enzyme of cholesterol biosynthesis
3-hydroxy-3-methyl glutaryl coenzyme A (HMG-CoA)
reductase, which catalyzes the reduction of HMG-CoA to
mevalonate during cholesterol biosynthesis (Alberts et al.
1980; Hajjaj et al. 2001). In addition to this lovastatin has a
clear evidence of benefit on stroke (Hebert et al. 1997) and
it also shows in vivo tumor suppression by inhibiting the
S. A. Sayyad : B. P. Panda (*) : M. Ali
Pharmaceutical Biotechnology Laboratory, Faculty of Pharmacy,
Jamia Hamdard (Hamdard University),
Hamdard Nagar,
New Delhi 110062, India
e-mail: bibhu_panda31@rediffmail.com
S. Javed
Molecular Biology Laboratory, Faculty of Science,
Jamia Hamdard (Hamdard University),
Hamdard Nagar,
New Delhi 110062, India

synthesis of nonsterol isoprenoid compounds (Valera et al.

2005). Lovastatin was the first statin to obtain approval from
the United States Food and Drug Administration in August
1987 (Tobert 2003, Demain 1999; Manzoni and Rollini 2002)
Lovastatin can be produced from numerous fungi, including
Monascus purpureus (Su et al. 2003; Wang et al. 2003).
Process optimization is a tedious process due to
involvement of multivariable process parameters. In this
process, screening of important factors is initially carried
out and these selected factors are then optimized by
different techniques (Box and Hunter 1957; Lewis et al.
1999). Response surface methodology (RSM) is a threefactorial design that gives relationship between one or more
measured dependent responses with a number of input
(independent) factors. RSM has some advantages that
includes less experiment numbers, suitability for multiple
factor experiments, search for relativity between factors,
and finding of the most suitable condition and forecast
response (Chang et al. 2006). In this, linear or quadratic
effects of experimental variables construct contour plots
and a model equation fitting the experimental data. This
facilitates the determination of optimum value of factors
under investigation and prediction of response under
optimized condition (Chakravarti and Sahai 2002).
The objective of the present study was to optimize the
nutrients for lovastatin production by M. purpureus MTCC
369 using shake flask method. RSM was used to optimize
key nutrients screened by PlackettBurman design.
Materials and methods
Microorganism
Culture of M. purpureus MTCC 369 was obtained from
Institute of Microbial Technology, Chandigarh, India,

Appl Microbiol Biotechnol (2007) 73:10541058

maintained on slants of potato-dextrose agar medium (Hi

Media, India) at 4C, and subcultured every 30 days.

1055
Table 1 Levels of nutrient parameters used in experiment
Nutrient parameter (g/l)

Preparation of seed culture

Spore suspension of M. purpureus MTCC 369 was
prepared from actively growing slants in sterile water and
diluted to a concentration of 5.7103. A 15% spore
suspension was inoculated to conical flasks containing the
basal medium [100 g dextrose, 10 g peptone, 2 g KNO3,
2 g NH4H2PO4, 0.5 g MgSO47H2O, and 0.1 g CaCl2 in
1,000 ml distilled water, adjusted to pH 6.0 (Su et al.
2003)]. These cultures were incubated at 30C for 48 h in a
shaker incubator at 110 rpm (Su et al. 2003).
Fermentation
All experiments were carried out in 250-ml Erlenmeyer
flasks containing 50 ml media as per experimental design
(Table 2). These flasks were inoculated with 10% seed
culture and incubated at 30C for 14 days on a rotary
shaker at 110 rpm (Su et al. 2003).

Dextrose
NH4Cl
KH2PO4
MgSO47H2O
MnSO4H2O

Levels
1

+1

5
1
1
0.2
0

22.5
3
2.5
1.1
0.25

40
5
4
2
0.5

Procedure given by Samiee et al. 2003 for HPLC

analysis was slightly modified. Lovastatin was estimated
by HPLC (SHIMADZU, Japan) using 2504.6 mm ID
Lichrosper 100 C18 column, 5 m particle size. Acetonitrile to water acidified (0.1%) with ortho-phosphoric acid
(65:35 v/v), was used as mobile phase. Flow rate of mobile
phase was maintained at 1.5 ml/min and detection was
carried out by a UV detector at 235 nm (Samiee et al.
2003).

Results
Design of experiment
Experimental design was formulated according to Box
Behnken tool of RSM using DESIGN EXPERT 7.0
software (Statease, USA) for selected five nutrient parameters. The various levels of nutrients were summarized in
Table 1. Relative effects of two variables on response were
identified from contour plots. An optimum value of the
factors for maximum production of lovastatin was determined by point prediction tool of the software.
Lovastatin estimation
Five milliliters of fermented medium was sonicated,
adjusted to pH 3.0 using 2N H3P04, and extracted with
ethyl acetate at ambient temperature. The mixture was
centrifuged at 3,000g for 8 min. Supernatant was
collected, lactonized with 1% trifluoroacetic acid, and
concentrated. Finally, acetonitrile was added for highperformance liquid chromatography (HPLC) analysis (Su
et al. 2003).

To identify the concentration of key nutrients influencing

lovastatin production, a shake flask method was used. Five
medium components (dextrose, NH 4 Cl, KH 2 PO 4 ,
MgSO47H2O, and MnSO4H2O) screened by Plackett
Berman design were chosen for study. An experimental
design of 46 runs containing 6 central points was made
according to BoxBehnkens response surface design for 5
selected parameters. The individual and interactive effects
of these nutrient variables were studied by conducting the
fermentation run at randomly selected different levels
(Table 1) of all five parameters. The response was measured
in terms of lovastatin production. The results of experimental data and simulated values are listed in Table 2. Data
collected for lovastatin concentration in each run were
analyzed using the software DESIGN EXPERT 7.0 and
fitted into a multiple nonlinear regression model proposes
following equation (in the coded factor) for lovastatin
production:

Lovastinmg=l 320:293 37:612A 3:966B  12:095C  23:431D  58:663E  26:268AB

40:019AC  62:277AD  65:07AE  88:116BC  33:796BD  48:926BE
15:383CD 17:649CE 7:301DE  121:783A2  89:115B2  70:906C 2
114:549D2  104:509E 2

1056

Appl Microbiol Biotechnol (2007) 73:10541058

Table 2 BoxBehnken design with results (actual and predicted)

Run

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46

Dextrose (g/l)

22.50
22.50
40.00
22.50
22.50
22.50
22.50
40.00
22.50
40.00
5.00
40.00
40.00
22.50
40.00
40.00
22.50
5.00
22.50
5.00
22.50
5.00
22.50
22.50
5.00
22.50
22.50
22.50
5.00
22.50
22.50
22.50
40.00
22.50
22.50
22.50
5.00
22.50
22.50
22.50
22.50
22.50
22.50
22.50
5.00
22.50

NH4Cl (g/l)

3.00
1.00
3.00
1.00
1.00
5.00
5.00
3.00
3.00
3.00
3.00
5.00
3.00
1.00
3.00
3.00
3.00
3.00
3.00
3.00
3.00
5.00
5.00
5.00
3.00
5.00
3.00
3.00
1.00
3.00
3.00
1.00
1.00
1.00
3.00
3.00
3.00
3.00
3.00
3.00
5.00
3.00
3.00
3.00
3.00
3.00

KH2PO4 (g/l)

2.50
2.50
4.00
2.50
4.00
2.50
2.50
2.50
1.00
2.50
2.50
2.50
2.50
2.50
2.50
1.00
1.00
2.50
2.50
2.50
1.00
2.50
2.50
2.50
4.00
1.00
2.50
2.50
2.50
2.50
2.50
2.50
2.50
1.00
4.00
1.00
1.00
2.50
4.00
2.50
4.00
4.00
2.50
2.50
2.50
4.00

where A, B, C, D, and E represent dextrose, NH4Cl,

KH2PO4, MgSO47H2O, and MnSO4H2O, respectively in
grams per liter.
The effects of all nutrient parameters on lovastatin
production can be compared with the help of a perturbation

MgSO47H2O (g/l)

2.00
1.10
1.10
0.20
1.10
1.10
0.20
2.00
1.10
1.10
1.10
1.10
1.10
1.10
0.20
1.10
2.00
0.20
1.10
1.10
1.10
1.10
1.10
2.00
1.10
1.10
0.20
1.10
1.10
1.10
1.10
2.00
1.10
1.10
1.10
0.20
1.10
1.10
2.00
2.00
1.10
0.20
0.20
1.10
2.00
1.10

MnSO4H2O (g/l)

0.00
0.00
0.25
0.25
0.25
0.00
0.25
0.25
0.50
0.50
0.00
0.25
0.00
0.50
0.25
0.25
0.25
0.25
0.25
0.50
0.00
0.25
0.50
0.25
0.25
0.25
0.00
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.50
0.25
0.25
0.25
0.25
0.50
0.25
0.25
0.50
0.25
0.25
0.00

Lovastatin (mg/l)
Actual

Predicted

156.62
101.69
96.19
128.99
241.63
256.67
158.44
14.70
55.98
13.39
37.72
104.20
278.29
96.64
232.34
247.82
112.65
57.59
319.87
33.10
208.24
97.02
55.78
41.92
96.53
279.12
162.26
320.05
29.92
319.33
320.54
147.66
143.90
103.77
119.72
136.30
88.08
320.97
66.64
56.62
64.52
151.82
33.05
321.01
89.07
201.39

129.17
132.40
113.10
102.30
232.33
238.26
177.82
35.86
80.66
7.88
49.98
124.28
255.35
113.00
207.28
217.33
138.88
7.50
320.29
62.80
233.29
102.45
23.01
63.37
117.92
264.45
190.63
320.29
41.12
320.29
320.29
123.03
169.74
80.28
91.77
154.98
62.07
320.29
83.93
26.44
64.03
161.56
58.70
320.29
85.20
173.80

plot (Fig. 1). The lines in the graph represent influence and
sensitivity of respective factor for lovastatin production.
This model resulted in ten response surface graphs. The few
response surface plots of calculated model for lovastatin
production are shown in Fig. 2(ad). The analysis of

Appl Microbiol Biotechnol (2007) 73:10541058

Fig. 1 Perturbation plot showing the effect of all nutrient parameter

on lovastatin production. A, B, C, D, and E represent dextrose, NH4Cl,
KH2PO4, MGSO47H2O, and MnSO4H2O, respectively

variance of regression for lovastatin production was

summarized in Table 3. In the case of lovastatin
production this calculated model is able to explain 94.9%
of the results.
All the response surfaces/contour could be analyzed for
determining the optimized value of the factors, but it was
difficult to analyze all these simultaneously. Hence, point
prediction of design expert software was used to determine
the optimum values of the factors for maximum lovastatin
production. Finally, the optimum values of dextrose
29.59 g/l, NH4Cl 3.86 g/l, KH2PO4 1.73 g/l, MgSO47H2O
0.86 g/l, and MnSO4H2O 0.19 g/l were determined. These
values predict 351 mg/l of lovastatin production. These
optimized values of nutrient parameters were validated in a
duplicate shake flask study and an average 326 mg/l of
lovastatin production was obtained. This shows 93%
validity of the predicted model.

Discussion
RSM proved to be a powerful tool for optimizing lovastatin
production by M. purpureus MTCC 369. From the studied
nutrient variables different ingredients have different
effects on lovastatin production. Dextrose and NH4Cl
were positively significant factors, whereas others were

Fig. 2 Response surface plot showing relative effect of two nutrient

parameters on lovastatin production while keeping others at constant
level

1057

1058

Appl Microbiol Biotechnol (2007) 73:10541058

Table 3 Analysis of variance of calculated model for lovastatin

production

References

Results of the analysis of variance

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Regression
Sum of squares
df
Mean squares
F ratio
P
Residual
Sum of squares
df
Mean squares
Correlation coefficient (R2)
Coefficient of variation (CV%)

407,875.19
20
20,393.76
23.4569
<0.0001
3,857.31
10
385.73
0.9494
20.18

Analysis of variance from DESIGN EXPERT 7.0

df Degree of freedom

negatively significant. The proposed model equation

illustrates the interaction between two factors. From
the equation it was found that KH 2 PO 4 and
MgSO47H2O interacted positively with MnSO4H2O,
whereas dextrose and NH4Cl showed negative interaction
with KH2PO4 and MgSO47H2O with respect to lovastatin
production. The relative effect of medium components on
lovastatin production while keeping other parameters
constant was depicted in the contour (two-dimensional)
and response surface (three-dimensional) graphs. From
Fig. 2ad it is evident that increase in dextrose concentration along with NH4Cl enhances lovastatin production
followed by a decrease in production. Remaining parameters, KH2PO4, MgSO47H2O, and MnSO4H2O gives
maximum lovastatin production at lower concentration
levels.
Fungus M. purpureus MTCC 369 produced 326 mg
lovastatin/l of optimized fermentation media, which is
much higher than that of lovastatin produced by M.
purpureus CCRC31615, Aspergillus terreus BST, and
Monascus paxii AM12M reported by Su et al. (2003) and
Manzoni et al. (1999) in submerged fermentation.