Hindawi Publishing Corporation

e Scientific World Journal

Volume 2014, Article ID 510627, 15 pages
http://dx.doi.org/10.1155/2014/510627

Research Article
Optimization of Large-Scale Culture
Conditions for the Production of Cordycepin with
Cordyceps militaris by Liquid Static Culture

Chao Kang,1,2 Ting-Chi Wen,1 Ji-Chuan Kang,1 Ze-Bing Meng,1

Guang-Rong Li,1 and Kevin D. Hyde3,4
1
The Engineering and Research Center of Southwest Bio-Pharmaceutical Resources, Ministry of Education,
Guizhou University, Guiyang, Guizhou 550025, China
2
Institute of Biology, Guizhou Academy of Sciences, Guiyang, Guizhou 550009, China
3
Institute of Excellence in Fungal Research, School of Science, Mae Fah Luang University, Chiang Rai 57100, Thailand
4
Botany and Microbiology Department, College of Science, King Saud University, Riyadh 11442, Saudi Arabia

Correspondence should be addressed to Ji-Chuan Kang; bcec.jckang@gzu.edu.cn

Received 14 February 2014; Accepted 8 April 2014; Published 23 June 2014

Academic Editor: Rajesh Jeewon

Copyright © 2014 Chao Kang et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Cordycepin is one of the most important bioactive compounds produced by species of Cordyceps sensu lato, but it is hard to produce
large amounts of this substance in industrial production. In this work, single factor design, Plackett-Burman design, and central
composite design were employed to establish the key factors and identify optimal culture conditions which improved cordycepin
production. Using these culture conditions, a maximum production of cordycepin was 2008.48 mg/L for 700 mL working volume
in the 1000 mL glass jars and total content of cordycepin reached 1405.94 mg/bottle. This method provides an effective way for
increasing the cordycepin production at a large scale. The strategies used in this study could have a wide application in other
fermentation processes.

1. Introduction Cordycepin (3󸀠 -deoxyadenosine), a nucleoside analog,

was first isolated from C. militaris [6] and is one of the species
Cordyceps militaris is an entomopathogenic fungus belonging most important biologically active metabolites. It has been
to Ascomycota, Sordariomycetidae, Hypocreales, and Cordy- regarded as a medicinal agent responsible for immunological
cipitaceae [1] and is one of the most important traditional regulation [7], anticancer [8], antifungus [9], antivirus [10],
Chinese medicinal mushrooms. Cordyceps militaris is the antileukemia [11, 12], and antihyperlipidemia [13] activities.
type species of Cordyceps, which internally parasitizes larva Cordycepin is also a Phase I/II clinical stage drug candidate
or pupa of lepidopteran insects and forms fruiting bodies for treatment of refractory acute lymphoblastic leukemia
on their insect hosts. Cordyceps militaris has long been rec- (ALL) patients who express the enzyme terminal deoxynu-
ognized as a desirable alternative for natural Ophiocordyceps cleotidyl transferase (TdT) (http://www.ClinicalTrials.Gov
sinensis [2] as it has been given Chinese Licence number verified by OncoVista, Inc., 2009).
Z20030034/35. This is because the gathering of Ophiocordy- In previous work, cordycepin has been synthesized by
ceps sinensis is causing substantial reductions in populations chemical [14, 15] and microbial fermentation using C. mili-
[3]. Cordyceps militaris produces many bioactive compounds, taris [6] or Aspergillus nidulans [16, 17]. Solid-state fermen-
including polysaccharides, cordycepin, adenosine, amino tation [18, 19], submerged culture [4, 20–24], and surface
acid, organic selenium, ergosterol, sterols, cordycepic acid, liquid culture [25–27] have been used in microbial fermenta-
superoxide dismutase (SOD), and multivitamins [4, 5]. tion of cordycepin. Cordycepin obtained through chemistry
2 The Scientific World Journal

pathways is hard to purify, and the cost is much higher glass jars for analyzing biomass dry weight and cordycepin
than through biology fermentation. Thus a major need is to production.
improve the biology methodology [28]. Fermentation time
is too long and is difficult to achieve large scale production 2.3. Static Culture Conditions. The effects of factors affect-
via solid-state fermentation [18, 19]. Productivity is generally ing cell growth and the production of cordycepin by C.
low, the costs are high, and fermentation processes are easily militaris were studied using a one-factor-at-a-time method
contaminated in submerged culture in large fermenters [4, 20, for static culture. The effects of carbon sources on cordy-
21, 29]. Productivity in surface culture techniques is higher cepinproduction were studied by substituting carbon sources
as compared to other methods [29, 30] and the cost is lower such as sucrose, lactose, soluble starch, and dextrin for
[23]. New technologies, such as space mutation treatment and glucose at 25∘ C for 35 days. Effects of nitrogen sources
high-energy ion beam irradiation, have been used to obtain (yeast extract, beef extract, NH4 NO3 , NaNO3 , NH4 Cl,
better Cordycepin producing, novel mutants of C. militaris. casein, and carbamide) and inorganic salts (MgCl2 ⋅6H2 O,
The resulting mutants were higher cordycepin produces, MgSO4 ⋅7H2 O, KCl, ZnSO4 , CaCl2 ⋅2H2 O, CaSO4 ⋅2H2 O,
than the wild strain [30, 31]. Bu et al. [20] reported that FeSO4 ⋅7H2 O, and K2 HPO4 ⋅3H2 O) were also studied using
the cordycepin in C. militaris was substantially increased by static culture. Growth factors (Vitamin B1 (VB1 ), Vita-
the elicitor of Phytophthora sp. Research result showed that min B6 (VB6 ), Vitamin B7 (VB7 ), Vitamin B11 (VB11 ), 𝛼-
glucose and yeast extract were effective media components naphthylacetic acid (NAA), 3-Indoleacetlc acid (IAA), and
for improved cordycepin production by C. militaris [32, 2,4-dichlorophenoxyacetic acid (2,4-D)) were supplemented
33]. There have been other studies using different culture for 10 mg/L in basal media. Nucleoside analogues (1 g/L) and
conditions [21, 24, 25, 32], culture medium, and additives amino acids (8 g/L) established in our previous study [23] as
[4, 22–24, 26, 27] for the production of cordycepin via liquid an initial concentration were separately added to the optimal
culture. However, as far as we know, these reports studied concentration of carbon and nitrogen source, inorganic salts,
cordycepin production in 250 mL or 500 mL Erlenmeyer and growth factors and cultivated at 25∘ C for 35 days. All
flasks, and there have been no reports to improved cordycepin experiments were carried out at triplicate, and mean of results
production using static liquid culture in 1000 mL glass jars. is presented.
The latter process is a good way to scale up large scale
cordycepin production from the laboratory to industry. 2.4. Analytical Methods. Samples collected at 35 days from
In this study, the effects of working volume, carbon the glass jars were centrifuged at 2810 ×g for 20 min. The
sources, nitrogen sources, inorganic salts, growth factor, mycelium at the bottom of tubes was washed sufficiently with
nucleoside analogue, and amino acid additions were studied a large amount of distilled water and dried to a constant dry
in order to improve the cordycepin production by static liquid weight at 55∘ C.
culture of C. militaris (strain CGMCC2459) in 1000 mL glass For analysis of extracellular cordycepin, the resulting
jars. The results suggested that the optimization medium culture filtrate was obtained by centrifugation at 2810 ×g
conditions were helpful for improved large scale cordycepin for 20 min. The supernatant was filtered through a 0.45 𝜇m
production. membrane and the filtrate was analyzed by HPLC (1100
series, Agilent Technology, USA). Accurate quantities of
cordycepin (Sigma, USA) were dissolved in distilled water,
2. Materials and Methods to give various concentrations for calibration. The mobile
2.1. Microorganism and Seed Culture. The isolate of C. mil- phase was 10 mmol/L KH2 PO4 , which was dissolved in
itaris (strain CGMCC2459) used in the present study was methanol/distilled water (6 : 94). Elution was performed at a
collected from Mt. Qingcheng in Sichuan Province, China. flow rate of 1.0 mL/min with column temperature at 45∘ C and
The microorganism was maintained on potato dextrose agar UV wavelength of 259 nm. Mean values were computed from
(PDA) slants. Slants were incubated at 25∘ C for 7 days and triplicate samples.
then stored at 4∘ C. The seed culture was grown in a 250 mL
flask containing 70 mL of basal medium (sucrose 20 g/L; pep- 2.5. Plackett-Burman Design. The Plackett-Burman design,
tone 20 g/L; KH2 PO4 1 g/L; and MgSO4 ⋅7H2 O 0.5 g/L) at 25∘ C an effective technique for medium-component optimization
on a rotary shaker incubator at 150 rev/min for 5 days [24]. [35, 36], was used to select factors that significantly influ-
enced hydrogen production. Sucrose (𝑋1 ), peptone (𝑋2 ),
2.2. Basal Medium and Static Culture of Glass Jars. The K2 HPO4 ⋅3H2 O (𝑋4 ), MgSO4 ⋅7H2 O (𝑋5 ), and VB1 (𝑋6 )
basal medium composition for the fermentation was as were investigated as key ingredients affecting cordycepin
follows: sucrose 20 g/L; peptone 20 g/L; KH2 PO4 1 g/L; and production. Based on the Plackett-Burman design, a 15-run
MgSO4 ⋅7H2 O 0.5 g/L. The pH was not adjusted, followed was applied to evaluate eleven factors (including two virtual
by autoclaving for 30 min on the 121∘ C. The static culture variables). Each factor was prepared in two levels: −1 for low
experiments were performed in 1000 mL glass jars (inner level and +1 for high level. Table 1 illustrates the variables
diameter 110 mm, height 150 mm) containing basal medium and their corresponding levels used in the experimental
after inoculating with 10% (v/v; the biomass dry weight of design. The values of two levels were set according to
seed culture is 54 mg/mL) of the seed culture. The culture was our preliminary experimental results. The Plackett-Burman
incubated at 25∘ C without moving for 35 days, and samples design and the response value of cordycepin production are
were collected at the end of the fermentation from the shown in Table 2.
The Scientific World Journal 3

Table 1: Range of different factors investigated with Plackett- 800

Burman design. 10
700

Total content of cordycepin (mg)

Cordycepin production (mg/L)
Experimental value
Symbol Variables 600 8
Low (−1) High (+1)

Biomass dry weight (g)

𝑋1 Sucrose (g/L) 20 25 500
𝑋2 Peptone (g/L) 20 25 6
400
𝑋3 Virtual 1 −1 1
𝑋4 K2 HPO4 ⋅3H2 O (g/L) 1 1.25 300 4
𝑋5 VB1 (g/L) 10 12.5 200
𝑋6 MgSO4 ⋅7H2 O (g/L) 1 1.25 2
100
𝑋7 Virtual 2 −1 1
0 0
100 200 300 400 500 600 700 800 900
Working volume (mL)
2.6. Response Surface Methodology. Response surface meth-
odology using a central composite design was applied to Cordycepin production
Total content of cordycepin
batch cultures of C. militaris, for identifying the effects of pro- Biomass dry weight
cess variables [35, 36]. In this study, the basic nutrient (carbon
sources, nitrogen sources, inorganic salts, and growth factors) Figure 1: Effects of working volume on the production of cordy-
and additives (amino acid, nucleoside analogue) were studied cepin, total production of cordycepin, and biomass dry weight
for cordycepin production using static liquid culture. In (total content ofcordycepin (mg) = cordycepin production (mg/L)
the first test, a three-factor, five-level central composite × working volume (mL)).
design with 20 runs was employed. Tested variables (sucrose,
K2 HPO4 ⋅3H2 O, and MgSO4 ⋅7H2 O) were denoted as 𝑋1 , 𝑋4 ,
and 𝑋6 , respectively, and each of them was assessed at five
In this study, we tried to establish the most efficient
different levels, combining factorial points (−1, −1), axial
working volume of medium for improved cordycepin pro-
points (−1.6818, +1.6818), and central point (0), as shown in
duction. Cultures of C. militaris were prepared at the working
Table 3. Based on the above results, another test, a three-
volumes of 100 to 900 mL (corresponding to a medium depth
factor, five-level central composite design with 20 runs was
of 1.26 to 11.31 cm). As shown in Figure 1, cordycepin pro-
employed. Tested variables (amino acid, nucleoside analogue,
duction reduced gradually with increasing working volume
and culture time) were denoted as 𝐴, 𝐵, and 𝐶, respectively,
of the medium, from 100 to 700 mL. However, there was no
and each of them was assessed at five different levels, com-
significant difference in cordycepin production in different
bining factorial points (−1, +1), axial points (−1.6818, +1.6818),
working volumes. Obviously, the highest working volume of
and central point (0), as shown in Table 4.
900 mL did not help in cordycepin production. The result
indicates that there is an upper dissolved oxygen limit in
2.7. Statistical Analysis. Dry weight and cordycepin produc- the medium for cordycepin production [21]. Lower working
tion are expressed as means ± SD. An analysis of variance volumes result in higher cordycepin productivity, with the
(ANOVA) followed by Tukey’s test was applied for multiple highest peak (463.33 ± 56.72 mg of cordycepin) produced
comparisons of significant analyses at 𝑃 < 0.05. Statistical at using 700 mL of media. Changes in biomass values were
data analyses were performed in SPSS version 17.0 software small (between 300 and 700 mL) because of the restricted area
packet. Design-Expert Version 8.0.5b software package (Stat- and thickness of the mycelial mat. In order to obtain higher
Ease Inc., Minneapolis, USA) was used for designing exper- cordycepin production, the most effective medium amount
iments as well as for regression and graphical analysis of the was 700 mL (corresponding to an 8.8 cm medium depth) and
experimental data obtained. used as the media volume for next experiment.

3. Results and Discussion 3.2. Effects of Carbon and Nitrogen Sources on Cordycepin
Production. To find a suitable carbon source for C. militaris
3.1. Effects of Working Volume on the Biomass and Cordycepin cordycepin production we added various carbon sources at
Production. Dissolved oxygen concentration is the key factor a concentration of 20 g/L to the sugar-free basal medium.
in the medium for cell growth and metabolite biosynthesis Glucose was previously found to be an excellent precur-
[21]. Dissolved oxygen does not only have an important sor of cordycepin production [39]. However, as shown in
function in the respiratory chain, but also in metabolite com- Figure 2(a), sucrose and lactose proved to be better carbon
position [37, 38]. A previous study showed that the highest sources for cordycepin production than glucose in this
cordycepin production and productivity were obtained at study. Cordycepin production reached 843.63 ± 66.70 mg/L
lower dissolved oxygen levels [21]. Masuda et al. [25] also of sucrose and 823.72 ± 85.64 mg/L of lactose, respectively.
reported that a lower medium depth was most efficient for Therefore, sucrose was selected as the main carbon source in
cordycepin production in C. militaris by surface culture. the remaining experiment.
4 The Scientific World Journal

Table 2: Plackett-Burman design and response values.

Experimental value
Runs 𝑌 (mg/L) Cordycepin production
𝑋1 𝑋2 𝑋3 𝑋4 𝑋5 𝑋6 𝑋7
1 1 −1 1 −1 −1 −1 1 812.36 ± 26.83
2 1 1 −1 1 −1 −1 −1 1395.18 ± 8.4
3 −1 1 1 −1 1 −1 −1 900.25 ± 10.29
4 1 −1 1 1 −1 1 −1 802.45 ± 45.43
5 1 1 −1 1 1 −1 1 1097.66 ± 25.57
6 1 1 1 −1 1 1 −1 845.87 ± 24.94
7 −1 1 1 1 −1 1 1 786.35 ± 7.61
8 −1 −1 1 1 1 −1 1 805.08 ± 29.1
9 −1 −1 −1 1 1 1 −1 920.48 ± 16.21
10 1 −1 −1 −1 1 1 1 694.01 ± 79.51
11 −1 1 −1 −1 −1 1 1 497.28 ± 4.44
12 −1 −1 −1 −1 −1 −1 −1 592.83 ± 16.13
13 0 0 0 0 0 0 0 1134.14 ± 2.59
14 0 0 0 0 0 0 0 1100.21 ± 0.08
15 0 0 0 0 0 0 0 1133.56 ± 1.85

Table 3: Factors and levels of central composite design for carbon Table 4: Factors and levels of central composite design for amino
sources and inorganic salts. acid, nucleoside analogue, and time.

Code level Code level

Symbol Variables Symbol Variables
−1.6818 −1 0 1 1.6818 −1.6818 −1 0 1 1.6818
𝑋1 Sucrose (g/L) 3.1821 10 20 30 36.8179 𝐴 Hypoxanthine (g/L) 0.53 1 5 9 10.53
𝑋4 K2 HPO4 ⋅3H2 O (g/L) 0.1591 0.5 1 1.5 1.8409 𝐵 L-alanine (g/L) 5.27 8 12 16 18.72
𝑋6 MgSO4 ⋅7H2 O (g/L) 0.1591 0.5 1 1.5 1.8409 𝐶 Culture time (days) −0.09 4 10 16 20.09

In previous work, nitrogen showed a regulating role highest cordycepin production (1120.30 ± 105.28 mg/L) by
important in cordycepin production and had two effects [40]. C. militaris was observed in medium, when K2 HPO4 ⋅3H2 O
One effect was negative since, in excess, N promoted a faster was used as an inorganic salt. KH2 PO4 , MgSO4 ⋅7H2 O,
mycelial growth and consequently diverted the source of KCl, and MgCl2 ⋅6H2 O were also useful inorganic salts. At
carbon toward energy and biomass production. The other last, MgSO4 ⋅7H2 O and K2 HPO4 ⋅3H2 O were recognized as
effect was positive because a moderate input contributed favorable bioelements for production of cordycepin.
to the maintenance of citric acid productive biomass [40]. Growth factor is essential for growth response and metab-
To investigate the effect of nitrogen sources on cordycepin olite production [42]. In order to find the optimal growth
production in C. militaris, various compounds containing factor for cordycepin production, C. militaris was cultured
nitrogen (inorganic and organic nitrogen) were added indi- in a basal medium with different vitamins and plant growth
vidually to nitrogen free basal medium at a concentration of hormones in static liquid culture. Cordycepin production
20 g/L. Among the 8 nitrogen sources tested, peptone, yeast increased in media with added 10 mg/L of VB1 , NAA,
extract, beef extract, casein, and NH4 NO3 were favorable and VB11 (Figure 3(b)). Maximum cordycepin production
to the cordycepin production (Figure 2(b)). Organic nitro- (1159.34 ± 109.01 mg/L) occurred when VB1 was used as the
gen was advantageous to both growth and biosynthesis of growth factor.
metabolites. The result is consistent with the experimental
data reported [18] and showed that maximum cordycepin
production resulted when the peptone was used as a nitrogen 3.4. Screening of Important Variables Using Plackett-Burman
source. Design. The data (Table 2) indicated wide variation in cordy-
cepin production in the 15 tests. The data suggested that pro-
3.3. Effects of Inorganic Salt and Growth Factor on the Cordy- cess optimization is important for improving the efficiency of
cepin Production. Inorganic ion was one of the most impor- cordycepin production. Analysis of the regression coefficients
tant nutrition components of medium for the mycelial growth and 𝑡 values of 7 factors (Table 5) showed that 𝑋1 , 𝑋2 , 𝑋4 ,
[41]. In order to investigate the effects of inorganic salt for the and 𝑋5 had positive effects on cordycepin production. 𝑋6
cordycepin production in C. militaris, we tested nine types (at had negative effects. The variable affects with a confidence
1 g/L) of inorganic salts (Figure 3(a)). Media with only 20 g/L level above 95% are considered as significant factors. Based on
glucose and 20 g/L peptone were used as the control. The these results, three factors (𝑋1 , sucrose; 𝑋4 , K2 HPO4 ⋅3H2 O;
The Scientific World Journal 5

∗∗ ∗∗
900 1000
∗∗
800 900
∗∗
800
Cordycepin production (mg/L)

Cordycepin production (mg/L)

700 ∗∗
700
600 ∗
600
500 ∗∗
500
400
400
300 #
300 ∗∗
200
200
100 100
0 0
Control Glucose Dextrin Sucrose Lactose Soluble

Control

Peptone

Yeast extract

Casein

NH4 Cl

NH4 NO3

NaNO3
Beef extract

Carbamide
starch
Carbon source

Cordycepin production
Nitrogen sources

Cordycepin production
(a) (b)

Figure 2: Effects of carbon sources and nitrogen sources on the production of cordycepin: carbon sources (a); nitrogen sources (b); ∗ 5%
significance level (test group versus control group); ∗∗ 1% significance level (test group versus control group); # 5% significance level (control
group versus test group).

∗∗
∗∗
1200 1200

∗∗
1000 1000 ∗
Cordycepin production (mg/L)
Cordycepin production (mg/L)

∗
800 ∗∗ ∗∗ 800
∗∗ ∗

600 600

400 400

200 200

0 0
Control 2,4-D
Control

K2 HPO4 ·3H2 O

MgCl2 ·6H2 O

MgSO4 ·7H2 O

CaSO4 ·2H2 O

CaCl2 ·2H2 O

KCl

FeSO4 ·7H2 O
KH2 PO4

IAA
ZnSO4

VC VB1 NAA VB11

Growth factor
Cordycepin production

Inorganic salts
Cordycepin production
(a) (b)

Figure 3: Effects of inorganic salt and growth factors on the production of cordycepin: inorganic salt (a); growth factors (b); ∗ 5% significance
level (test group versus control group); ∗∗ 1% significance level (test group versus control group).
6 The Scientific World Journal

Table 5: Results of regression analysis of Plackett-Burman design.

Regression analysis
Symbol
Effect Coefficient Standard error 𝑇 𝑃
845.82 32.86 25.74 0.000∗∗
𝑋1 190.88 95.44 32.86 2.90 0.027∗
𝑋2 149.23 74.62 32.86 2.27 0.064
𝑋3 −40.85 −20.42 32.86 −0.62 0.557
𝑋4 244.10 122.05 32.86 3.71 0.010∗
𝑋5 62.81 31.41 32.86 0.96 0.376
𝑋6 −176.15 −88.08 32.86 −2.68 0.037∗
𝑋7 −127.39 −63.69 32.86 −1.94 0.101
Ct Pt 276.82 73.48 3.77 0.009∗∗
∗
5% significance level; ∗∗ 1% significance level; 𝑋1 –𝑋7 are symbols shown in Table 1.

Table 6: Experimental design and responses of the central composite design for carbon sources and inorganic salts.

Variables Code Variables Code

Run 𝑌 (mg/L) Cordycepin production Run 𝑌 (mg/L) Cordycepin production
𝑋1 𝑋4 𝑋6 𝑋1 𝑋4 𝑋6
1 −1 −1 −1 1080.55 ± 109.69 11 0 0 0 1399.43 ± 124.44
2 1 −1 −1 1359.48 ± 12.61 12 0 0 0 1415.43 ± 42.13
3 −1 1 −1 1158.59 ± 12.15 13 0 0 0 1487.42 ± 16.38
4 1 1 −1 1289.90 ± 47.00 14 0 0 0 1409.43 ± 155.22
5 −1 −1 1 980.55 ± 34.72 15 −1.6818 0 0 876.91 ± 16.69
6 1 −1 1 1097.48 ± 52.42 16 1.6818 0 0 1290.00 ± 14.71
7 −1 1 1 987.95 ± 2.89 17 0 −1.6818 0 1110.57 ± 157.63
8 1 1 1 1117.38 ± 116.84 18 0 1.6818 0 1344.66 ± 65.54
9 0 0 0 1485.38 ± 12.19 19 0 0 −1.6818 958.39 ± 224.14
10 0 0 0 1333.48 ± 94.22 20 0 0 1.6818 958.00 ± 75.82

and 𝑋6 , MgSO4 ⋅7H2 O) were considered as significant for model was significant. In this case, linear terms of 𝑋1 and
cordycepin production by static liquid culture methodology. quadratic terms of 𝑋12 , 𝑋42 , 𝑋62 were significant of model terms
for cordycepin production. The “Lack of Fit F value” of 0.0903
3.5. Optimization by Response Surface Methodology for Car- implied that the “Lack of Fit” was not significant relative to
bon Sources and Inorganic Salts. In order to evaluate the the pure error (𝑃 > 0.05). The Pred-𝑅2 of 0.3183 was not as
influence of medium component on cordycepin production, close to the Adj-𝑅2 of 0.7954 as one might normally expect.
sucrose, K2 HPO4 ⋅3H2 O, and MgSO4 ⋅7H2 O should be exam- The result suggested that some factors were not considered in
ined. The levels of variables for central composite design the model. However, the “Adeq Precision” of 8.173 indicated
experiments were selected according to the above results of that the model was adequate for prediction production of
Plackett-Burman design. Table 6 shows the detailed experi- cordycepin.
mental design and results. Regression analysis was performed The response surface plot obtained from (1) is shown in
to fit the response function (cordycepin production) with Figure 4. It is evident that cordycepin production reached
the experimental data. From the variables obtained (Table 6), its maximum at a combination of coded level (𝑋1 , sucrose,
the model is expressed by (1), which represents cordycepin level 0.47; 𝑋4 , K2 HPO4 ⋅3H2 O, level 0.21; 𝑋6 , MgSO4 ⋅7H2 O,
production (𝑌1 ) as a function of sucrose (𝑋1 ), K2 HPO4 ⋅3H2 O level −0.20) when using canonical analysis of the Design-
(𝑋4 ), and MgSO4 ⋅7H2 O (𝑋6 ) concentrations: Expert Version 8.0.5b software package. The model predicted
a maximum response of 1451.43 mg/L cordycepin production
𝑌1 = 1419.68 − 98.95𝑋1 + 31.45𝑋4 − 51.68𝑋6 at levels of sucrose 24.7 g/L, K2 HPO4 ⋅3H2 O 1.11 g/L, and
MgSO4 ⋅7H2 O 0.90 g/L as optimized medium components.
− 16.89𝑋1 𝑋4 − 20.48𝑋1 𝑋6 + 2.36𝑋4 𝑋6 (1)

− 106.03𝑋12 − 55.06𝑋42 − 150.31𝑋62 . 3.6. Effects of Nucleoside Analogue and Amino Acid on
the Production of Cordycepin. Chassy and Suhadolnik [43]
Results of 𝐹-test analysis of variance (ANOVA) showed reported that adenine and adenosine were precursors for
that the regression was statistically significant at 95% and 99% cordycepin synthesis. Amino acids were regarded as the best
confidence levels (Table 7). The “𝐹 value” of the model was substance for improved cordycepin production [4, 23]. Based
9.21, and the value of “Prob > 𝐹” < 0.01 indicated that the on these results, among 10 different kinds of nucleoside
The Scientific World Journal 7

Table 7: ANOVA for response surface quadratic polynomial model for carbon sources and inorganic salts.

Source Sum of quares df Mean Square 𝐹-value 𝑃-value Prob > 𝐹

Model 6.507𝐸 + 005 9 72300.77 9.21 0.0009∗∗
𝑋1 -𝑋1 1.337𝐸 + 005 1 1.337𝐸 + 005 17.03 0.0021∗∗
𝑋4 -𝑋4 13504.30 1 13504.30 1.72 0.2190
𝑋6 -𝑋6 36477.48 1 36477.48 4.65 0.0565
𝑋1 𝑋4 2282.55 1 2282.55 0.29 0.6016
𝑋1 𝑋6 3356.89 1 3356.89 0.43 0.5279
𝑋4 𝑋6 44.38 1 44.38 5.653𝐸 − 003 0.9416
𝑋1 2 1.620𝐸 + 005 1 1.620𝐸 + 005 20.63 0.0011∗∗
𝑋4 2 43685.18 1 43685.18 5.56 0.0400∗∗
𝑋6 2 3.256𝐸 + 005 1 3.256𝐸 + 005 41.47 <0.0001∗∗
Residual 78513.73 10 7851.37
Lack of Fit 61670.32 5 12334.06 3.66 0.0903
Pure Error 16843.41 5 3368.68
Cor Total 7.292𝐸 + 005 19
𝑅2 = 0.8923; CV = 7.34%; Pred-𝑅2 = 0.3183; Adj-𝑅2 = 0.7954; Adeq Precision = 8.173; ∗ 5% significance level; ∗∗ 1% significance level.

Table 8: Experimental design and responses of the central composite design for amino acid, nucleoside analogue, and time.

Variables Code Variables Code

Run 𝑌 (mg/L) Cordycepin production Run 𝑌 (mg/L) Cordycepin production
𝐴 𝐵 𝐶 𝐴 𝐵 𝐶
1 −1 −1 −1 1383.01 ± 41.53 11 0 0 0 2041.25 ± 54.70
2 1 −1 −1 1422.52 ± 39.41 12 0 0 0 2020.97 ± 73.70
3 −1 1 −1 1216.88 ± 8.69 13 0 0 0 1998.18 ± 49.48
4 1 1 −1 1857.51 ± 164.86 14 0 0 0 2068.60 ± 72.79
5 −1 −1 1 1216.87 ± 253.38 15 −1.6818 0 0 1590.14 ± 222.14
6 1 −1 1 1111.18 ± 170.50 16 1.6818 0 0 1573.90 ± 776.16
7 −1 1 1 1536.05 ± 75.17 17 0 −1.6818 0 1636.44 ± 65.23
8 1 1 1 851.70 ± 17.01 18 0 1.6818 0 1527.98 ± 177.46
9 0 0 0 2073.27 ± 65.85 19 0 0 −1.6818 1211.14 ± 82.58
10 0 0 0 1743.09 ± 14.81 20 0 0 1.6818 676.97 ± 142.74

analogue were supplemented for 1 g/L in this study. As shown the significant factors (hypoxanthine, L-alanine, and culture
in Figure 5(a), cordycepin production increased obviously time) and their optimal levels. Figure 6 shows the morpho-
in the medium with hypoxanthine, thymine, and thymi- logical characteristics of C. militaris in 1000 mL glass jars after
dine additives. The highest production of cordycepin was fermentation by static liquid fermentation. Table 8 shows the
achieved, when hypoxanthine was used as the nucleoside detailed experimental design and results. Regression analysis
analogue. Hypoxanthine’s molecular structure is similar to was performed to fit the response function (cordycepin
purine bases found in cordycepin. Substituent on purine production) with the experimental data. From the variables
bases structure is –OH on hypoxanthine rather than –NH2 . obtained (Table 9), the model was expressed by (2), which
The –OH should be replaced in metabolic pathways. In represented cordycepin production (𝑌2 ) as a function of
addition, among 14 different amino acids were tested for hypoxanthine (𝐴), L-alanine (𝐵), and culture time (𝐶, time),
8 g/L. As shown in Figure 5(b), L-alanine can improve cordy- concentrations:
cepin production. Previous research showed that adenine,
adenosine, and glycine were good additives for increased 𝑌2 = 1991.13 − 10.05𝐴 + 10.70𝐵 − 151.02𝐶
cordycepin production [4, 23, 26, 27]. L-alanine may be an + 2.81𝐴𝐵 − 183.77𝐴𝐶 − 26.14𝐵𝐶 (2)
important nutritional element for C. militaris or component
of cordycepin production. Hypoxanthine and L-alanine were − 146.09𝐴2 − 146.03𝐵2 − 371.65𝐶2 .
the best additives to promote cordycepin production in this
study. Results of 𝐹-test analysis of variance (ANOVA) showed
that the regression was statistically significant at 95% and
3.7. Optimization by Response Surface Methodology for Amino 99% confidence level (Table 9). The “𝐹 value” of the model
Acid, Nucleoside Analogue, and Fermentation Time. Simi- was 11.91, and the value of “Prob > 𝐹” < 0.01 indicated
larly, central composite design was also applied to study that the model was significant. In this case, linear terms
8 The Scientific World Journal

Cordycepin production (mg/L)

1.50
1400
1500
Cordycepin production (mg/L)

1.30
1400
Prediction 1451.43

K2 HPO4 ·3H2 O (g/L)

1300
1.10
1200
1300
1100 0.90 1400

1.50 30.00 0.70 1200

1.30 25.00
K H 1.10 20.00
2 P 0.90 L)
O
4 ·3H 0.70 15.00 e (g/
s 0.50
2O 0.50 10.00 cro
(g/ Su 10.00 15.00 20.00 25.00 30.00
L)
Sucrose (g/L)
Design-Expert Software
Factor Coding: Actual Design-Expert Software
Cordycepin production Factor Coding: Actual
Design points above predicted Cordycepin production
Design points below predicted Design points
1487.42 1487.42
876.911 876.911
X1 = A: sucrose Actual factor X1 = A: sucrose Actual factor
X2 = B: K2 HPO4 C: MgSO4 = 1.00 X2 = B: K2 HPO4 C: MgSO4 = 1.00

(a)
Cordycepin production (mg/L)
1.50 1100
1500 1200
Cordycepin production (mg/L)

1400 1.30
1300
1300
MgSO 4 ·7H2 O (g/L)

1200 1.10

1100 1400
1000 0.90
Prediction 1451.43

1.50 30.00 0.70

1.30 25.00 1200
1.10 20.00
Mg 0.90 L)
SO
4 ·7H 0.70 15.00 e (g/
s 0.50
2O 0.50 10.00 cro
(g/ Su 10.00 15.00 20.00 25.00 30.00
L)
Sucrose (g/L)
Design-Expert Software
Factor Coding: Actual Design-Expert Software
Cordycepin production Factor Coding: Actual
Design points above predicted Cordycepin production
Design points below predicted Design points
1487.42 1487.42

876.911 876.911
X1 = A: sucrose Actual factor X1 = A: sucrose Actual factor
X2 = C: MgSO4 B: K2 HPO4 = 1.00 X2 = C: MgSO4 B: K2 HPO4 = 1.00

(b)

Figure 4: Continued.
The Scientific World Journal 9

Cordycepin production (mg/L)

1.50
1200
1500 1300
Cordycepin production (mg/L)

1400 1.30

1300

MgSO 4 ·7H2 O (g/L)

1200 1.10

1100
Prediction 1451.43
1000 0.90

1400
1.50 1.50 0.70
1.30 1.30
Mg 1.10 1.10 ) 1300
SO 0.90 0.90 g/L
4 ·7H
0.70 O(
2O 0.70 ·3H 2
0.50
(g/ 0.50 0.50 O4
L)
2
HP 10.00 15.00 20.00 25.00 30.00
K
K2 HPO4 ·3H2 O (g/L)
Design-Expert Software
Factor Coding: Actual Design-Expert Software
Cordycepin production Factor Coding: Actual
Design points above predicted Cordycepin production
Design points below predicted Design points
1487.42 1487.42

876.911 876.911

X1 = B: K2 HPO4 Actual factor X1 = B: K2 HPO4 Actual factor

X2 = C: MgSO4 A: sucrose = 20.00 X2 = C: MgSO4 A: sucrose = 20.00

(c)

Figure 4: Three-dimensional response surface plots and two-dimensional contour plots for cordycepin production by C. militaris (strain
CGMCC2459) showing variable interactions of (a) sucrose and K2 HPO4 ⋅3H2 O; (b) sucrose and MgSO4 ⋅7H2 O; (c) K2 HPO4 ⋅3H2 O and
MgSO4 ⋅7H2 O.

Table 9: ANOVA for response surface quadratic polynomial model for amino acid, nucleoside analogue, and time.

Source Sum of quares df Mean Square 𝐹-value 𝑃-value Prob > 𝐹

Model 2.899𝐸 + 006 9 3.221𝐸 + 005 11.91 0.0003∗∗
𝐴-𝐴 1378.39 1 1378.39 0.051 0.8260
𝐵-𝐵 1564.06 1 1564.06 0.058 0.8148
𝐶-𝐶 3.115𝐸 + 005 1 3.115𝐸 + 005 11.51 0.0068∗
𝐴𝐵 63.06 1 63.06 2.331𝐸 − 003 0.9624
𝐴𝐶 2.702𝐸 + 005 1 2.702𝐸 + 005 9.99 0.0102∗
𝐵𝐶 5468.49 1 5468.49 0.20 0.6626
𝐴2 3.076𝐸 + 005 1 3.076𝐸 + 005 11.37 0.0071∗∗
𝐵2 3.073𝐸 + 005 1 3.073𝐸 + 005 11.36 0.0071∗∗
𝐶2 1.990𝐸 + 006 1 1.990𝐸 + 006 73.58 <0.0001∗∗
Residual 2.705𝐸 + 005 10 27053.63
Lack of Fit 1.928𝐸 + 005 5 38561.98 2.48 0.1707
Pure Error 77726.41 5 15545.28
Cor Total 3.169𝐸 + 006 19
𝑅2 = 0.9146; CV = 10.70%; Pred-𝑅2 = 0.4162; Adj-𝑅2 = 0.8378; Adeq Precision = 11.222; ∗ 5% significance level; ∗∗ 1% significance level.
10 The Scientific World Journal

2000 ∗∗
1800
∗

Cordycepin production (mg/L)

2000
∗∗ 1600
1800 ∗ ∗ 1400
Cordycepin production (mg/L)

1600
1400 1200
# #
1200 ## 1000 ## ## ##
# ## ##
1000 800
800
600
600
400
400
200 200

0 0

Aspartic acid
Control

L-hydroxyproline
L-arginine
L-glutamine
L-proline
Lysine
Glycine
L-glutamic acid
L-methionine
L-alanine
Asparagine
Cyteine
L-valine
L-isoleucine
Control

Adenine

Adenosine

Uridine

Hypoxanthine

Thymine

Thymidine

Cytosine

Guanine
Cytidine

Guanosine
Nucleoside analogue
Amino acids
Cordycepin production
Cordycepin production
(a) (b)

Figure 5: Effects of nucleoside analogue and amino acid on the production of cordycepin: nucleoside analogue (a); amino acid (b); ∗ 5%
significance level (test group versus control group); ∗∗ 1% significance level (test group versus control group); # 5% significance level (control
group versus test group); ## 1% significance level (control group versus test group).

of 𝐶; interactive terms of 𝐴𝐶; and quadratic terms of 𝐴2 , enhance the productivity of cordycepin and biomass in C.
𝐵2 , and 𝐶2 were significant in model terms for cordycepin militaris.
production. The “Lack of Fit F value” of 0.1707 implied that
the “Lack of Fit” was not significant relative to the pure error 3.8. Verification Experiments and Batch Culture. Based on
(𝑃 > 0.05). The Pred-𝑅2 of 0.4162 was not as close to the the results of response surface methodology, the optimized
Adj-𝑅2 of 0.8378 as one might normally expect. The result medium was prepared as follows: peptone 20 g/L; sucrose
suggested that some factors were not also considered in the 24.7 g/L; K2 HPO4 ⋅3H2 O 1.11 g/L; MgSO4 ⋅7H2 O 0.90 g/L;
model. However, the “Adeq Precision” of 11.222 indicated VB1 10 mg/L; hypoxanthine 5.45 g/L; and L-alanine 12.23 g/L.
that the model was adequate for prediction production of Five experiments were performed to confirm the above
cordycepin. optimal culture requirements. The data were 2011.15 mg/L,
The response surface plot obtained from (2) is shown in 2000.69 mg/L, 1989.22 mg/L, 1969.6 mg/L, and 2061.37 mg/L,
Figure 7. It is evident that cordycepin production reached respectively. The average cordycepin production was
its maximum with a combination of coded level (𝐴, hypox- 2006.41 ± 34.37 mg/L. The experimental values were
anthine, level 0.11; 𝐵, L-alanine, level 0.06; 𝐶, time, level particularly close to the predicted values (2008.48 mg/L).
−0.23) by canonical analysis of the Design-Expert Version The result confirmed the model suited the predictive of
8.0.5b software package. The model predicted a maximum hyperproduction of cordycepin by C. militaris in static liquid
response of 2008.48 mg/L cordycepin production at levels culture. Batch culture was carried for cordycepin production
of hypoxanthine 5.45 g/L, L-alanine 12.23 g/L, and time 8.6 under optimized culture conditions (Figure 8).
days (in the practical test 8 days) as optimized medium
components. 3.9. In Vitro Cordycepin Production Using Liquid Culture in
In previous work, the orthogonal design method [44– Other Studies. The highest report for cordycepin production
46], Box-Behnken design [34, 47], and central composite was 14300 mg/L by Masuda et al. [29] (Table 10). In our
design [31] were used to optimize culture conditions for experiment, cordycepin production at 2008.48 mg/L was
cordycepin production by Cordyceps sp. These experimental lower. However, a maximum total content of cordycepin
designs have been successfully used to optimize medium for (1405.94 mg) was achieved in our study. This is a second
the mycelial growth and microbial metabolite production in higher report of cordycepin production in one single fer-
liquid culture processes. In this study, static liquid culture menter. The results showed that the culture conditions will
conditions are optimized for the cordycepin production using provide an effective way for increasing cordycepin produc-
response surface methodology and are an effective way to tion.
The Scientific World Journal 11

Table 10: Cordycepin production in the medium by liquid culture in different studies.

Working volume of Total content of

Cordycepin
No. Methodology the medium cordycepin in one References
production (mg/L)
v/v (mL/mL) bottle (mg)
1 Submerged culture 50/250 245.7 12.5 Mao et al., [32]
Mao and Zhong
2 Submerged culture 50/250 420.5 21.03
[21]
3 Surface liquid culture 100/500 640 64 Masuda et al., [25]
4 Shaking + Static 100/250 2214.5 221.45 Shih et al., [34]
5 Surface liquid culture 100/500 2500 250 Masuda et al., [26]
6 Surface liquid culture 100/500 3100 310 Das et al., [30]
7 Surface liquid culture 100/500 8570 857 Das et al., [27]
8 Submerged culture 100/500 1644.21 164.42 Wen et al., [23]
9 Dark + Shaking 100/500 1015 101.5 Kang et al., [24]
10 Surface liquid culture 150/500 14300 2145 Masuda et al., [29]
11 Static liquid culture 700/1000 2008.48 1405.94 In this study

Figure 6: Morphology of C. militaris (strain CGMCC2459) in 700/1000 mL glass jars at the end of the fermentation process by response
surface methodology: symbols in photos indicated 20 runs.
12 The Scientific World Journal

Cordycepin production (mg/L)

16.00
1800 1800
2010
Cordycepin production (mg/L)

1757.5 14.00
Prediction 2008.48
1505

L-Alanine (g/L)
1252.5 12.00

1000

10.00
16.00 9.00
14.00 7.00 1900 1800
1800
5.00 /L)
L-A 12.00 e (g
lan 3.00 hin
ine 10.00 ant 8.00
(g/ 8.00 1.00 pox
L) Hy 1.00 3.00 5.00 7.00 9.00
Design-Expert Software Hypoxanthine (g/L)
Factor Coding: Actual Design-Expert Software
Cordycepin production Factor Coding: Actual
Design points above predicted Cordycepin production
Design points below predicted Design points
2073.27 2073.27

676.97 676.97
X1 = A: Hypoxanthine X1 = A: Hypoxanthine Actual factor
Actual factor
X2 = B: L-alanine C: time = 10.00 X2 = B: L-alanine C: time = 10.00

(a)
Cordycepin production (mg/L)
16.00
1400
2010 1600
Cordycepin production (mg/L)

1757.5 1800
13.00

1505
Time (days)

1252.5 10.00
Prediction 2008.48
1000
2000

7.00
16.00 9.00
13.00 7.00 1800
5.00 /L)
10.00
in e (g 1600
Tim 3.00 h
e (d 7.00 ant
4.00 pox 4.00
ays
) 1.00 Hy 1.00 3.00 5.00 7.00 9.00
Design-Expert Software Hypoxanthine (g/L)
Factor Coding: Actual Design-Expert Software
Cordycepin production Factor coding: Actual
Design points above predicted Cordycepin production
Design points below predicted Design points
2073.27 2073.27
676.97 676.97
X1 = A: Hypoxanthine Actual factor X1 = A: Hypoxanthine Actual factor
X2 = C : Time B : L-Alanine = 12.00 X2 = C : Time B : L-Alanine = 12.00
(b)

Figure 7: Continued.
The Scientific World Journal 13

Cordycepin production (mg/L)

16.00
1600
2010
Cordycepin production (mg/L)

1757.5 1800
13.00

1505

Time (days)
1252.5 10.00
Prediction 2008.48
1000
2000

7.00
16.00 16.00
13.00 14.00
10.00 12.00 1600
L) 1800
Tim 10.00 (g/
e (d 7.00 n i ne 4.00
ays
) 4.00 8.00
L- Ala 8.00 10.00 12.00 14.00 16.00
Design-Expert Software L-Alanine (g/L)
Factor Coding: Actual
Cordycepin production Design-Expert Software
Design points above predicted Factor Coding: Actual
Design points below predicted Cordycepin production
Design points
2073.27
2073.27
676.97
676.97
X1 = A: Hypoxanthine Actual factor X1 = A: Hypoxanthine Actual factor
X2 = C: Time A: Hypoxanthine = 5.00 X2 = C: Time A: Hypoxanthine = 5.00
(c)

Figure 7: Three-dimensional response surface plots and two-dimensional contour plots for cordycepin production by C. militaris (strain
CGMCC2459) showing variable interactions of (a) hypoxanthine and L-alanine; (b) hypoxanthine and time; (c) L-alanine and time.

Figure 8: Batch culture for cordycepin production under optimized culture conditions by static liquid culture using C. militaris (strain
CGMCC2459).

4. Conclusion 0.90 g/L; VB1 10 mg/L; hypoxanthine 5.45 g/L; and L-alanine
12.23 g/L. Hypoxanthine and L-alanine were added to the
In this work, single factor design, Plackett-Burman design, optimal medium at 8.6 days. Optimal incubation conditions
and central composite design were employed to establish were 25∘ C at an unaltered pH of 35 days. Using these
the key factors and identify optimal culture conditions culture conditions, a maximum production of cordycepin
which improved cordycepin production by C. militaris was 2008.48 mg/L for 700 mL working volume in the
CGMCC2459. Optimal media contained peptone 20 g/L; 1000 mL glass jars, and total content of cordycepin reached
sucrose 24.7 g/L; K2 HPO4 ⋅3H2 O 1.11 g/L; MgSO4 ⋅7H2 O 1405.94 mg/bottle (700 mL/1000 mL). This method provides
14 The Scientific World Journal

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