Process Biochemistry 40 (2005) 1667–1672

www.elsevier.com/locate/procbio

Optimization of carbon source and carbon/nitrogen ratio for

cordycepin production by submerged cultivation of
medicinal mushroom Cordyceps militaris
Xian-Bing Maoa, Titiporn Eksriwongb, Somchai Chauvatcharinb, Jian-Jiang Zhonga,*
a
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China
b
Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
Received 9 February 2004; accepted 11 June 2004

Abstract

Effects of various carbon sources and carbon/nitrogen ratios on production of a useful bioactive metabolite, cordycepin (30 -
deoxyadenosine), by submerged cultivation of a Chinese traditional medicinal mushroom Cordyceps militaris were investigated in shake
flasks. The carbon sources examined were lactose, sucrose, glucose, fructose, galactose, maltose and xylose, and glucose was found to be
most favourable to cordycepin production, whereas cells grew best in galactose medium. The dry cell weight (DW) was increased with an
increase in initial glucose concentration within the range of 25–70 g/l as investigated. The highest cordycepin production, i.e. 245.7 
4.4 mg/l on day 18, was obtained in medium containing 40 g glucose/l. To enhance further the cordycepin production, the effect of
carbon/nitrogen ratios was studied using central composite design and response surface analysis. The maximum cordycepin production
and productivity of 345.4  8.5 mg/l and 19.2  0.5 mg/l per day were achieved in medium with optimized carbon and nitrogen sources,
i.e. 42.0 g glucose/l and 15.8 g peptone/l. The information obtained is helpful for the hyperproduction of cordycepin by submerged
cultivation of C. militaris on a large scale.
# 2004 Elsevier Ltd. All rights reserved.

Keywords: Medicinal mushroom; Submerged cultivation; Cordycepin; Cordyceps militaris; Response surface analysis; Central composite design

1. Introduction
Mushroom is an abundant source of a wide range of
useful natural products with biological activities. Since the
field cultivation of mushroom takes several months to yield
the fruiting body with a low productivity of bioactive
compounds, submerged cultivation of mushrooms is viewed
as a promising alterative for producing valuable substances
[1–4]. However, there have been few investigations on the
bioprocess development of mushroom submerged fermenta-
tion [1,2].
Cordyceps militaris, a caterpillar-shaped Chinese tradi-
tional medicinal mushroom, is an entomopathogenic fungus,
which belongs to the class Ascomycetes and DongChong-
XiaCao group in Chinese herbs [5]. Besides its usage as a
* Corresponding author. Tel.: +86-21-64252091; fax: +86-21-64253904.
E-mail address: jjzhong@ecust.edu.cn (J.-J. Zhong).
crude drug, it has been extensively used as folk tonic food or
invigorant since ancient times [5,6]. This mushroom
produces an important bioactive compound, cordycepin
(30 -deoxyadenosine), which is a nucleoside analogue [7,8].
Cordycepin is reported to possess many interesting
biological and pharmacological activities, including immu-
nological stimulating, anti-cancer, anti-virus and anti-
infection activities [7–11]. Previous work reported the
isolation of cordycepin from liquid culture medium of C.
militaris and its pharmacological functions [7,8,12–13].
Trace levels of cordycepin were mentioned in mycelium and
culture broth during submerged cultivation of Cordyceps sp.
in potato-dextrose medium [14]. Jia et al. reported that
7.1 mg cordycepin/l was acquired in an airlift bioreactor
cultivation of C. militaris [15]. However, there have been no
reports on dynamic profiles of cordycepin production by
submerged cultivation of C. militaris, and the effect of
medium nutrients has not been revealed as yet.

0032-9592/$ – see front matter # 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2004.06.046
1668 X.-B. Mao et al. / Process Biochemistry 40 (2005) 1667–1672
Usually, culture medium is important to the yield of any
fermentation products, and carbon and nitrogen sources
generally play a significant role because these nutrients are
directly linked with cell proliferation and metabolite
biosynthesis [1–4,16]. Various statistical experimental
design strategies were applied to the optimization of
fermentation media such as for lovastatin production by
Monascus rubber [17]. But, as far as we know, there is
limited knowledge about the nutritional requirement for
cordycepin production by C. militaris, and there have been
no reports on medium optimization to improve cordycepin
production. In this work, the effects of carbon sources
and carbon/nitrogen ratios were focused in order to improve
the cordycepin production by submerged cultivation of
C. militaris. The information obtained is considered
fundamental and useful to the development of C. militaris
cultivation process for efficient production of cordycepin on
a large scale.
2. Materials and methods
2.1. Maintenance and seed culture of C. militaris
The strain of C. militaris was purchased from the
collection bank of Huazhong Agricultural University
(Hubei, China). The stock culture was maintained on
potato-dextrose-agar slants. The slants were inoculated with
mycelia and incubated at 25 8C for 7 days, and then used for
seed culture inoculation. The seed culture medium consisted
of the following components: glucose, 40 g/l; yeast extract,
10 g/l; KH2PO4, 0.5 g/l; K2HPO43H2O, 0.5 g/l and
MgSO47H2O, 0.5 g/l. The mycelia of C. militaris were
transferred to the seed culture medium by punching out
about 5 mm2 of the slants with a sterilized cutter. The seed
culture was grown in a 250 ml shake flask containing 50 ml
of liquid medium and incubated at 25 8C on a rotary shaker
(110 rpm) for 5 days.
2.2. Experiments on carbon source, initial glucose level
and carbon/nitrogen ratio
Effects of carbon sources on liquid culture of C. militaris
were studied using 40 g/l of one of the following carbon
sources, i.e. lactose, sucrose, glucose, fructose, galactose,
maltose and xylose. The other culture medium components
were 10 g/l of peptone, 0.5 g/l of KH2PO4, 0.5 g/l of
K2HPO43H2O and 0.5 g/l of MgSO47H2O. For the
investigation on initial sugar concentrations, glucose was
used with levels of 25, 40, 55 and 70 g/l tested. In
experiments on effects of carbon/nitrogen ratios, the levels
of glucose and peptone in medium were changed and a
statistical approach was applied. Inoculation was done by
transferring 5 ml of the above seed culture medium (with
ca. 12 g DW/l) to 45 ml medium in a 250 ml shake flask. The
cultivation was conducted at 25 8C on a rotary shaker
(110 rpm). In all experiments, multiple flasks at least in
duplicate were run at the same time to ensure reproduci-
bility.
2.3. Central composite design
Central composite design (CCD) was conducted in the
optimum vicinity to locate the true optimum concentrations
of glucose and peptone for cordycepin production [17]. The
levels of variables for CCD experiments were selected
according to the results of one-at-a-time strategy. The CCD
experimental results were fitted with a second-order
polynomial equation of Eq. (1) by a multiple regression
technique.
X X X
y ¼ b0 þ bi x i þ bii x2i þ bij xi xj (1)
where y is predicted response, b is constant coefficient, and x
is the coded independent variable.
The fitness of the second-order model was expressed by
the regression coefficient R2 and its statistical significance
was determined by an F-test. The regression significance
was tested by a t-test. The SAS software (version 8.0 by SAS
Institute Inc., NC, USA) and MatLab software (version 6.5
by the Mathworks, Inc.) were used for regression and
graphical analyses of the data obtained, respectively.
2.4. Analytical procedures
2.4.1. Determination of dry cell weight (DW), pH and
residual medium sugar
For the measurement of DW, the cells from a sample were
filtered through a filter paper and washed twice with distilled
water. The fresh cells were dried at 60 8C for sufficient time
until a constant DW was obtained. After sampling, one part
of the culture filtrate was used for measuring pH value with a
pH meter; another part was stored at 20 8C, and later
thawed for analyses of residual sugar and cordycepin
production. Residual sugar concentration was assayed by a
phenol–sulphuric acid method [18].
Average growth rate was calculated as: (maximum DW –
initial DW)/[(initial DW)(culture time)] (unit: day1), and
growth yield on sugar was calculated as: (maximum DW –
initial DW)/(initial sugar concentration – residual sugar
concentration)(unit: g DW/g sugar).
2.4.2. Cordycepin production [8,15]
Standard cordycepin (from Sigma) was dissolved in
distilled water for calibration. The cordycepin concentration
was analyzed by HPLC. A column Kromasil C18 (4.6 mm 
250 mm, 5 mm particle size) (Eliter Company, Liaonin,
China) was used. The mobile phase was 10 mM KH2PO4,
which was dissolved in methanol/distilled water (15:85) and
the mobile phase was driven by double pump (model: Waters
150, Millipore, USA). Elution was performed at a flow rate
of 1 ml/min with column temperature at 30 8C. The UV
 X.-B. Mao et al. / Process Biochemistry 40 (2005) 1667–1672 1669

by submerged cultivation of C. militaris, different carbohy-

Fig. 1. Effects of carbon sources on the cell growth by DW (A), sugar
consumption (B), medium pH (C) and cordycepin production (D) during
submerged cultivation of C. militaris. Symbols for different sugars: lactose
(&), sucrose (), glucose (~), fructose (&), galactose (*), maltose (~)
and xylose (*). The error bars in the figure indicate the standard derivations
from three independent samples.
wavelength of 254 nm was monitored by a tunable absorb-
ance detector (model: Waters 486, Millipore, USA). Syringe
of 50 ml was used for injection. Samples from Eppendorf
tubes were thawed and then centrifuged at 15,000  g for
15 min. The sample supernatant was used for cordycepin
concentration assay.
drates, i.e. lactose, sucrose, glucose, fructose, galactose,
maltose and xylose, were used. Other carbon sources
including mannitol, soluble starch and rapeseed oil were also
tested in preliminary experiments and a low cordycepin
production was obtained (data not shown).
The time profiles of cell growth, sugar consumption, pH
value and cordycepin production under various carbon
sources are compared in Fig. 1. A maximal cell density,
growth rate and cell yield were obtained in galactose
medium after 9 days of cultivation, which reached 19.27 
0.37 g DW/l, 1.58  0.03 d1 and 0.55 g DW/g sugar,
respectively. Except that in lactose medium, the residual
sugar was exhausted at the end of cultivation (on day 21). In
lactose medium, a low cell density (8.00  0.48 g/l) and
growth rate (0.25  0.02 d1) were observed.
The maximal cordycepin production titre was 68.5  7.0,
145.3  10.3, 262.7  6.7, 89.1  8.3, 39.7  2.1, 106.0 
3.9 and 19.7  2.8 mg/l in lactose, sucrose, glucose,
fructose, galactose, maltose and xylose medium, respec-
tively, and their corresponding productivity was 3.26 
0.33, 8.07  0.57, 14.59  0.37, 4.95  0.46, 2.20  0.12,
5.05  0.19 and 0.94  0.13 mg/l per day. From the above
data, it is apparent that the highest cordycepin production
and productivity were obtained in glucose medium, whereas
the cell growth in galactose medium was preferred. It is
possible that different carbon sources might have different
effects of catabolic repression on the cellular secondary
metabolism.
3.2. Effect of initial glucose concentrations

3. Results and discussion
3.1. Effect of carbon sources
Carbohydrates are important carbon and energy sources
for cultured cells. Effects of various carbon sources on
polysaccharide production by C. militaris were reported
[2,3], but only glucose was used as carbon source in
cordycepin production by the strain [7,12–15]. In order to
identify a suitable carbon source for cordycepin production
Based on the above results, glucose was selected as a
suitable carbon source for further studies. Table 1 sum-
marizes the effect of initial glucose concentrations on cell
growth (by the maximum DW) and cordycepin production.
Cell growth increased in parallel with an increase of initial
glucose concentrations. The average growth rate kept almost
constant (i.e. 0.93–1.06 d1), when the initial glucose
concentration was more than 25 g/l. Such a phenomenon
was also claimed in submerged cultivation of Ganoderma
lucidum [1]. The cell yield on sugar decreased with an

Table 1
Effects of initial glucose concentrations on cell growth and cordycepin production by submerged cultures of C. militaris
Parameters Initial glucose concentrations (g/l)
25 40 55 70
Maximum DW (g/l) 11.02  0.14a (day 6)b 16.30  0.34 (day 12) 17.85  0.07 (day 15) 21.88  0.25 (day 18)
Average growth rate (d1) 1.38  0.02 1.06  0.02 0.93  0.00 0.97  0.01
Cell growth yield on sugar (g DW/g sugar) 0.41 0.39 0.32 0.31
Maximum cordycepin production titre (mg/l) 158.9  0.3 (day 18)b 245.7  4.4 (day 18) 151.9  8.4 (day 12) 139.1  6.3 (day 15)
Cordycepin productivity (mg/l per day) 8.83  0.02 13.65  0.25 12.66  0.70 9.27  0.42
Cordycepin yield on cells (mg/g) 14.4 15.1 8.5 6.4
a
The maximum errors were calculated from two independent samples.
b
Cultivation time when the maximum DW or maximum cordycepin production was achieved.
1670 X.-B. Mao et al. / Process Biochemistry 40 (2005) 1667–1672

Table 2
Experimental design and responses (actual and predicted) of the central composite design (CCD)
Runs x1 (g/l) Glucose x2 (g/l) Peptone y (mg/l) Cordycepin production DW (g/l) observed
Observed Predicted
1 50 (+1) 20 (+1) 285.2  15.5a 295.4 23.0  0.2
2 50 (+1) 10 (1) 213.0  9.7 217.5 20.4  0.2
3 30 (1) 20 (+1) 230.2  14.5 221.5 16.6  0.3
4 30 (1) 10 (1) 264.8  5.6 250.5 14.3  0.1
5 54.14 (+1.414) 15 (0) 289.3  14.3 278.1 23.0  0.2
6 25.86 (1.414) 15 (0) 233.8  10.9 249.2 14.2  0.2
7 40 (0) 22.07 (+1.414) 248.1  19.3 246.2 20.0  0.2
8 40 (0) 7.93 (1.414) 205.4  5.8 211.5 17.1  0.3
9b 40 (0) 15 (0) 333.5 337.8 18.9
10 40 (0) 15 (0) 339.8 337.8 18.6
11 40 (0) 40 (0) 345.3 337.8 19.2
12 40 (0) 40 (0) 328.3 337.8 19.4
13 40 (0) 40 (0) 342.1 337.8 18.5
a
Samples were taken on day 18 and the standard deviation was calculated from three independent samples.
b
Runs of 9, 10, 11, 12 and 13 were replicates at the center point.

increase of initial glucose concentration (Table 1). For
cordycepin formation, its highest production and produc-
tivity titres were obtained at 40 g/l initial glucose. The
results indicate that a high initial glucose concentration (55
or 70 g/l) was unfavourable to cordycepin biosynthesis.
Here, the osmotic pressure caused by a high glucose
concentration may be detrimental to the metabolite
biosynthesis although the cell growth was not inhibited.
In ganoderic acid biosynthesis by Ganoderma lucidum [4]
and in butyric acid production by Clostridium populeti [19],
the inhibitory effect of osmotic pressure caused by a
relatively high initial sugar concentration on the metabolite
biosynthesis was claimed. Another possible reason is the
carbon catabolite repression caused by glucose as reported
in Saccharomyces cerevisiae [20–22].
3.3. Effect of carbon/nitrogen ratio
production (data not shown). Because the concentrations
of both carbon and nitrogen sources and their balance in
medium are very important for metabolite optimal produc-
tion, in the following the combined effect of carbon source
(glucose) and nitrogen source (peptone) was investigated
using the statistical approach of Box–Wilson central
composite design (CCD), which can help to identify and
quantify the interaction between variables [17].
The levels of variables for CCD experiments were
selected according to the above results of one-at-a-time
strategy. Table 2 shows the detailed experimental design and
results. Regression analysis was performed to fit the
response function (cordycepin production) with the experi-
mental data. From the variables obtained (Table 3), the
model was expressed by Eq. (2), which represented
cordycepin production (y) as a function of glucose (x1)
and peptone (x2) concentrations.

To increase cordycepin production further not only the yðmg=lÞ ¼ 337:8 þ 10:2x1 þ 12:2x2 þ 26:7x1 x2
carbon source but also the nitrogen source should be  37:1x21  54:5x22 (2)
examined. Various organic sources including yeast extract,
casein enzymatic hydrolysate, casein acid hydrolysate, Results of F-test analysis of variance (ANOVA) showed that
urea and peptone, and various inorganic sources, such as the regression was statistically significant (P < 0.05) at 95%
nitrate potassium, ammonia, ammonium nitrate and other of confidence level (Table 4). The model presented a high
ammonium salts were tested. Among all nitrogen sources regression coefficient (R2 = 0.9697).
examined, peptone was most beneficial to cordycepin The response surface plot obtained from Eq. (2) is shown
in Fig. 2. It is evident that cordycepin production reached its
Table 3 maximum at a combination of coded level 0.195 (x1,
Regression results from the data of central composite design (CCD)
experiments
glucose) and 0.160 (x2, peptone) by canonical analysis of
Parameters Parameter estimate Standard error T value Pr > jtj
Table 4
Intercept 337.799495 5.334526 63.32 <0.0001 ANOVA results for cordycepin production obtained from CCD
x1 10.2112 4.217631 2.42 0.0460
Regression DF Type I sum of squares R-square F value Pr > F
x2 12.249075 4.217631 2.90 0.0228
x1x1 37.091377 4.523544 8.20 <0.0001 Linear 2 2034.15486 0.0618 7.15 0.0204
x2x1 26.700000 5.964181 4.48 0.0029 Quadratic 2 27014 0.8212 94.93 <0.0001
x2x2 54.496633 4.523544 12.05 <0.0001 Crossproduct 1 2851.56000 0.0867 20.04 0.0029
Total model 5 31899 0.9697 44.84 <0.0001
x1: glucose concentration; x2: peptone concentration.
 X.-B. Mao et al. / Process Biochemistry 40 (2005) 1667–1672
Fig. 2. Response surface plot showing the combined effect of glucose and
peptone on cordycepin production by C. militaris.
SAS software. The model predicted a maximum response of
339.8 mg cordycepin/l at levels of 42.0 g glucose/l and
15.8 g peptone/l as optimized medium components.
Although the cell density could be increased with an
increase of glucose or peptone concentrations as investi-
gated (Tables 1 and 2), cordycepin production was inhibited
by high concentrations of glucose and peptone. Peptone
consists of amino acid, peptide and protein, and peptide and
protein may be decomposed by extracellular proteases into
amino acids, which in turn are consumed by mycelia [23]. In
general, different nitrogen compounds present different
effects on cell growth and secondary metabolite biosynthesis
[24]. A further study with a chemically defined medium is
required to understand which nitrogen compound in peptone
is beneficial to cordycepin production.
3.4. Cordycepin production in optimized medium
To confirm the above prediction, further experiments
using both optimized (as predicted) and non-optimized
media were performed. Similar data as shown in Fig. 1 were
Fig. 3. Time profile of cell growth by DW (&), glucose consumption (&),
medium pH (~) and cordycepin production (~) during submerged cultiva-
tion of C. militaris in the optimized medium with 42.0 g glucose/l and
15.8 g peptone/l. The error bars in the figure indicate the standard deriva-
tions from three independent samples.
1671
obtained in non-optimized medium (as control) with 40 g
glucose/l and 10 g peptone/l (data not shown). Fig. 3 shows
the time profiles of cell growth, sugar consumption, pH and
cordycepin production in the medium with optimized carbon
and nitrogen sources (as predicted). A maximal cell density
of 17.12  0.15 g DW/l was attained on day 9 and residual
glucose was almost exhausted at that time. A maximal
cordycepin production and productivity reached 345.4 
8.5 mg/l and 19.2  0.5 mg/l per day on day 18, which were
much higher than those of control. The medium pH quickly
decreased to about 2.5 and later maintained constant, which
was lower than that of control (Fig. 3 versus Fig. 1). Whether
a low pH value was beneficial to cordycepin production by
the cells is under investigation in this laboratory.
4. Conclusion
In this work, a process of submerged cultivation of C.
militaris for production of a bioactive compound, cordy-
cepin, was demonstrated. The effects of major nutrients, i.e.
carbon sources and carbon/nitrogen ratios, on cordycepin
production were studied in order to obtain a suitable
fermentation medium. Glucose was an optimal carbon
source for cordycepin production, although cell growth was
the best in galactose medium. According to the central
composite design and response surface analysis, an optimal
carbon source (42.0 g glucose/l) and nitrogen source (15.8 g
peptone/l) were identified and a maximal cordycepin
production (345.4  8.5 mg/l) and productivity (19.2 
0.5 mg/l per day) were successfully obtained. The funda-
mental information obtained in this work is beneficial for
further development of C. militaris cultivation process for
hyperproduction of cordycepin on a bioreactor scale. Such
work may also be helpful to other mushroom fermentations
for useful metabolite production.
Acknowledgement
Financial support from the National Science Fund
for Distinguished Young Scholars (NSFC project no.
20225619), the Cheung Kong Scholars Program adminis-
tered by the Ministry of Education of China, and the
Shanghai Leading Academic Discipline Program are
gratefully acknowledged.
References
[1] Tang YJ, Zhong JJ. Fed–batch fermentation of Ganoderma lucidum
for hyperproduction of polysaccharide and ganoderic acids. Enzyme
Microbial Technol 2002;31:20–8.
[2] Kim SW, Xu CP, Hwang HJ, Choi JW, Kim CW, Yun JW. Production
and characterization of exopolysaccharides from an enthomopatho-
genic fungus Cordyceps militaris NG3. Biotechnol Prog 2003;19:
428–35.
1672 X.-B. Mao et al. / Process Biochemistry 40 (2005) 1667–1672
[3] Park JP, Kim SW, Hwang HJ, Yun JW. Optimization of submerged
culture conditions for the mycelial growth and exo-biopolymer pro-
duction by Cordyceps militaris. Lett App Microb 2001;33:76–81.
[4] Fang QH, Zhong JJ. Submerged fermentation of higher fungus
Ganoderma lucidum for production of valuable bioactive metabo-
lites—ganoderic acid and polysaccharide. Biochem Eng J 2002;
10:61–5.
[5] Ying J, Mao X, Ma Q, Zong Y, Wen H. Icons of Medicinal Mushroom
from China. Beijing: Science Press; 1987 (in Chinese).
[6] Wu ZL, Wang XX, Chen WY. Inhibitory effect of Cordyceps sinensis
and Cordyceps militaris on human glomerular mesangial cell prolif-
eration induced by native LDL. Cell Biochem funct 2000;18:93–7.
[7] Cunningham KG, Hutchinson SA, Manson W, Spring FS. Cordycepin,
a metabolic product from cultures of Cordyceps militaris (Linn.) Link.
Part I. Isolation and characterisation. J Chem Soc 1951;2299–300.
[8] Ahn YJ, Park SJ, Lee SG, Shin SC, Choi DH. Cordycepin: selective
growth inhibitor derived from liquid culture of Cordyceps militaris
against Clostridium spp. J Agric Food Chem 2000;48:2744–8.
[9] Zhou XX, Meyer CU, Schmidtke P, Zepp F. Effect of cordycepin on
interleukin-10 production of human peripheral blood mononuclear
cells. Eur J Pharmacol 2002;453:309–17.
[10] De Julian-Ortiz JV, Galvez J, Munoz-Collado C, Garcia-Domenech R,
Gimeno-Cardona C. Virtual combinatorial syntheses and computa-
tional screening of new potential anti-herpes compounds. J Med Chem
1999;17:3308–14.
[11] Sugar AM, Mccaffrey RP. Antifungal activity of 30 -deoxyadenosine
(cordycepin). Antimicrob Agents Chemother 1998;42:1424–7.
[12] Frederiksen S, Malling H, Lenow H. Isolation of 30 -deoxyadenosine
(cordycepin) from the liquid medium of Cordyceps militaris (L. ex Fr.)
link. Biochem Biophys Acta 1965;95:189–93.
[13] Melling J, Belton FC, Kitching D, Stones WR. Production of pure
cordycepin (30 -deoxyadenosine) from Cordyceps militaris. J Pharm
Pharmacol 1972;24(suppl.):125.
[14] Hsu TH, Shiao LH, Hsieh C, Chang DM. A comparison of the
chemical composition and bioactive ingredients of the Chinese med-
icinal mushroom DongChongXiaCao, its counterfeit and mimic, and
fermented mycelium of Cordyceps sinensis. Food Chem 2002;78:
463–9.
[15] Jia JM, Zhang JX, Wu LJ. A method of cultivating and extracting
cordycepin by Cordyceps militaris in airlift bioreactor. CN Patent No.
1335389A, 2002. (in Chinese)
[16] Casas López JL, Sánchez Pérez JA, Fernández Sevilla JM, Acién
Fernández FG, Molina Grima E, Chisti Y. Production of lovastatin by
Aspergillus terreus: effects of the C:N ratio and the principal nutrients
on growth and metabolite production. Enzyme Microbial Technol
2003;33:270–7.
[17] Chang YN, Huang JC, Lee CC, Shih IL, Tzeng YM. Use of response
surface methodology to optimize culture medium for production of
lovastatin by Monascus rubber. Enzyme Microbial Technol 2002;
30:889–94.
[18] Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F. Colorimetric
method for determination of sugars and related substances. Anal Chem
1956;28:350–6.
[19] Patel GB, Agnew BJ. Growth and butyric acid production by Clos-
tridium populeti. Arch Microbiol 1988;150:267–71.
[20] Sankaran L, Pogell BM. Biosynthesis of puromycin in Strepto-
myces alboniger: regulation and properties of O-demethypuro-
mycin O-methyltransferase. Antimicrob Agents Chemother 1975;8:
721–32.
[21] Escalante L, Ramos I, Imriskova I, Langley E, Sanchez S. Glucose
repression of anthracycline formation in Streptomyces peucetius var.
caesius. Appl Microbiol Biotechnol 1999;52:572–8.
[22] Belinchón MM, Gancedo JM. Xylose and some non-sugar carbon
sources cause catabolite repression in Saccharomyces cerevisiae. Arch
Microbiol 2003;180:293–7.
[23] Zhu Y, Rinzema A, Bonarius HPJ. Tramper J, Bol J. Microbial
transglutaminase production by Streptoverticillium mobaraense: ana-
lysis of amino acid metabolism using mass balances. Enzyme Micro-
bial Technol 1998;23:216–26.
[24] Voelker F, Altaba S. Nitrogen source governs the patterns of growth
and pristinamycin production in Streptomyces pristinaespiralis.
Microbiology 2001;147:2447–59.