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Efficient production of cordycepin by the Cordyceps militaris mutant G81-3 for practical use
Process Biochemistry 49 (2014) 181–187
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Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Efficient production of cordycepin by the Cordyceps militaris mutant G81-3 for practical use Mina Masuda a , Shonkor Kumar Das a , Masanori Hatashita b , Shinya Fujihara a , Akihiko Sakurai a,∗ a b
Department of Applied Chemistry and Biotechnology, Graduate School of Engineering, University of Fukui, 3-9-1 Bunkyo, Fukui 910-8507, Japan Research & Development Department, The Wakasa-wan Research Center, 64-52-1 Nagatani, Tsuruga 914-0192, Japan
a r t i c l e
i n f o
Article history: Received 17 July 2013 Received in revised form 17 October 2013 Accepted 24 October 2013 Available online 1 November 2013 Keywords: Cordycepin Cordyceps militaris mutant Liquid surface culture Scale-up Crystallization
a b s t r a c t Cordycepin is one of the most versatile metabolites of Cordyceps militaris. When C. militaris G81-3, the mutant obtained by a proton beam irradiation, was cultivated by liquid surface culture, cordycepin was found to crystallize in the medium due to high cordycepin concentration. Because the cordycepin crystals strongly attached to the mycelial mat, complete recovery of cordycepin was difficult. To prevent cordycepin crystallization, increase of the initial medium volume was examined to decrease the condensation rate by vaporization, in combination with decrease of the initial substrate level. Besides, addition of the water to the culture bottle was examined to cancel out the medium condensation. A 7.4 g/L of cordycepin was obtained without crystallization by the former method. The upper limit of cordycepin production, 14.3 g/L, was obtained by the latter method and this value was the highest level of all the previous reports. A 2.5-fold scale-up of the culture slightly declined the production rate, however, the amount of cordycepin production was properly 2.5 times higher than that of the small scale. In addition, differences in sensitivities to the substrates between the mutant and the wild-type strain are discussed. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Medicinal mushrooms have been widely used for the promotion of health and for the prevention and treatment of various diseases due to their biological active constituents [1]. Among them, the fungi of the genus Cordyceps (belonging to Clavicipitaceae, Ascomycetes), which parasitize insects, are considered to be the more promising medicinal mushrooms [2]. Cordyceps sinensis and Cordyceps militaris, two well-known Cordyceps species have multiple physiological and pharmacological activities [3–8] derived from a variety of chemical components such as cordycepin, ergosterol, peptides, polysaccharides, and superoxide dismutase [9–17]. Cordycepin, a nucleoside analog (3 -deoxyadenosine), is the major active constituent of C. militaris and was first isolated from a culture broth of C. militaris in the 1950s [9]. Since then, many biological functions of cordycepin including anti-tumor, anti-metastatic, anti-bacterial, anti-viral, immunomodulatory and anti-inflammatory activities have been discovered [18–26]. In the
∗ Corresponding author. Tel.: +81 776 27 8924; fax: +81 776 27 8747. E-mail address: a [email protected] (A. Sakurai). 1359-5113/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.procbio.2013.10.017
last decade, cordycepin has been studied as a therapeutic agent for a variety of cancers, especially leukemia (ClinicalTrials.gov, Verified by OncoVista, Inc., 2009), trypanosomiasis and restenosis [27–30]. The designs of derivatives were also studied to protect against rapid in vivo oxidation [31]. These studies imply the need for the largescale production of cordycepin. As for the cordycepin production by C. militaris, the liquid surface culture [32], the submerged culture [33], and the combination of submerged and static cultures [34] were investigated to efficiently produce cordycepin. Recently, an efficient method to extract cordycepin from the waste mushroom beds was proposed [35]. In order to improve cordycepin production to a commercial level, a novel mutant was obtained from C. militaris using high-energy proton beam irradiation as reported in our previous study [36]. In the liquid surface culture of this mutant (G81-3), the concentrations of the carbon and nitrogen sources were optimized, and the peak cordycepin production was 2.8 times that of the wild-type strain [37]. In addition, the repeated batch culture method was investigated to achieve a higher production rate [g/(L d)] of cordycepin [38]. In this study, the culture conditions previously reported for G813 [37] was further investigated from the viewpoint of both timeand cost-savings by the liquid surface culture. The ultimate purpose of this research is the abundant supply of cordycepin in order to discover a novel pharmacological use and to advance the clinical trials of this compound.
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2. Materials and methods 2.1. Microorganisms and culture
2.2. Analytical methods The concentration of cordycepin was determined by an HPLC equipped with a UV detector (LC-9A system, Shimadzu Corp., Japan) and a reverse phase column (TSK-gel ODS-80Ts, Tosoh Corp., Japan). The mobile phase consisted of methanol and 0.1% (v/v) phosphoric acid (2/98, v/v). The flow rate was 1.0 mL/min, and the column temperature was 40 ◦ C. The chromatogram was monitored by the UV absorbance at 260 nm. The authentic standard was purchased from Sigma–Aldrich Co. The glucose concentration was measured by the mutarotase-GOD (glucose oxidase) method using a Glucose CII Test Wako (Wako Pure Chemical Industries, Ltd., Japan). The amino acid concentration was determined by measuring the absorbance at 420 nm of the reaction product of the primary amino groups with sodium 2, 4, 6-trinitrobenzenesulfonate (TNBS·Na) in the presence of sodium sulfite at pH 9.9 [40]. Since the sensitivity of proteins was much lower than that of the free amino acids, the detected value could be considered as that derived from the free amino acids. l-glycine was used for preparing the calibration curve. To determine the dry cell weight (DCW), the entire contents of the culture bottle were centrifuged (20 min at 15,000 × g). The separated mycelia were sufficiently washed with distilled water and freeze-dried for 48 h, then, the weight was measured. To measure the content of cordycepin in the crystallized compounds during the culture, all the insoluble particles, including the particles precipitated in the liquid medium and the particles attached to the mycelial mat, were collected and dried out on the filter paper. The attached particles were collected one by one with the tweezers. They were dissolved in the heated water (40 ◦ C) and the suspended solid were removed by filtration, then the filtrate was freeze-dried. After measurement of weight of all the freeze-dried products, it was dissolved in distilled water at 100 mg/L and the cordycepin concentration was determined by the HPLC. The cordycepin content in the crystallized compounds was calculated based on the cordycepin concentration of the said solution. To determine the volume of the evaporated water from the liquid medium, total weight of the culture bottle was measured periodically and the decrease in the weight was regarded as the volume of the water evaporation. All of the cordycepin concentrations shown in this experiment were corrected values considering the condensation or dilution of the medium due to vaporization or the addition of water. These values were obtained by dividing the total produced cordycepin (g) by the initial medium volume (L).
Cordycepin [g/L]
C. militaris NBRC 9787 and the mutant G81-3 [36] were used in the present experiments. The wild-type strain was stored on a potato dextrose agar (PDA) (Nissui Pharmaceutical Co., Ltd., Japan) slant, and the mutant was on an agar slant of Vogel’s medium [39] supplemented with 200 mM of 8-azaguanine at 5 ◦ C. Prior to the experiment, a piece of mycelia in the stock culture was transferred to a fresh PDA slant and incubated at 25 ◦ C for eight days. The seed culture was started by transferring one loopful of mycelia from each active slant to a PDA plate and incubated at 25 ◦ C for 13 days for the wild-type strain and for 20 days for the mutant. The inoculum was prepared by punching out a 1-cm disk from the PDA plate with a sterilized cylindrical cutter. The liquid surface culture was started by inoculating two seed disks into a 500-mL culture bottle, or five seed disks into a 1250-mL culture bottle. The diameter of the former bottle was 8.5 cm and that of the latter one was 13.5 cm. The heights of both were 14.0 cm with bottlenecks of 4.5 cm diameter and 4.0 cm height. The initial volume of the liquid medium was set between 100 mL and 300 mL (corresponding to 1.8 cm and 5.3 cm of medium depth) for the small bottle and was 378 mL (corresponding to 2.6 cm of medium depth) for the large one. The medium volume decreased during the culture by water evaporation from the liquid surface. In contrast, it increased when sterile distilled water was supplemented to avoid the medium condensation. In the latter case, a supporter (a stainless-steel round frame) was hung in the bottle in order to prevent the mycelial mat from soaking in the liquid medium. The bottleneck was filled with a cotton plug during the culture. The prepared bottles were placed in an incubator maintained at a temperature of 25 ± 1 ◦ C and a humidity of 30 ± 2%. Just before sampling, the medium was gently agitated for 5 s by a magnetic stirrer. A 1-mL portion of the medium was withdrawn through a sampling port typically at a 3-day interval and was filtered through a 0.45-m membrane filter in order to remove the suspended mycelia. The filtrate was analyzed for cordycepin, glucose and pH. The standard media for the wild-type strain and the mutant are as follows. For the wild-type strain, the medium (Medium W) was composed of 62.6 g/L of glucose and 72.5 g/L of yeast extract (Difco); for the mutant, the medium (Medium M) was composed of 86.2 g/L of glucose and 93.8 g/L of yeast extract. Both media contained Vogel’s medium diluted to a 1/10 concentration. These standard media had been determined in a previous investigation using the response surface method (RSM) to obtain the optimum concentration of the carbon and nitrogen sources for the cordycepin production. To check the reproducibility, the experiments were carried out at least triplicate.
Mutant Autoclave Mutant Filtration Wild-type Autoclave Wild-type Filtration
10 8 6 4 2 0 0
5
10
15
20
25
30
35
40
45
Cultrure time [d] Fig. 1. Comparison of time courses of cordycepin production by the wild-type strain and G81-3 using the filtered medium and the autoclaved medium. Initial medium volume: 100 mL.
3. Results and discussion 3.1. Effects of the medium sterilization process on cordycepin production by C. militaris or its mutant In the previous investigation using the RSM, the optimum medium concentration was extremely high. Nevertheless, the media had been sterilized by autoclave in the overall experiments. Therefore, the deactivation of vitamins and synthesis of unknown compounds by the Maillard reaction during autoclave might have some adverse effects on the cordycepin production. In order to determine the effect of the autoclave sterilization, the cordycepin production using the autoclaved medium was compared to that using the filtered one. Fig. 1 shows the time courses of the cordycepin production using the autoclaved medium and the filtered medium. (In all the figures, the symbols show the average values and the error bars show the minimum and maximum values.) Regardless of the sterilization method, the culture periods and final cordycepin productions were almost the same for the wild-type strain. On the other hand, for the mutant G81-3, the time courses showed significant differences between the sterilization methods. The use of the filtered medium significantly reduced the lag phase. The prolonged lag phase with the autoclaved medium was considered to be due to some inhibiting effect of unknown compounds synthesized by the autoclave on the growth of G81-3, since the cell growth and the glucose consumption were hardly observed before 15 d of culture time. In addition, the filtered medium increased cordycepin production and caused crystallization after 24 d. The content of cordycepin in the crystallized compounds was over 97%, and therefore the dissolved cordycepin concentration decreased due to the transfer of cordycepin from the liquid medium to the crystallized compounds. The produced cordycepin in the medium had kept oversaturation for a while (the solubility of cordycepin in water is approximately 7 g/L at 25 ◦ C), however, the locally-concentrated cordycepin around the mycelial mat started to crystallize as shown in the white circle of Fig. 2a. Cordycepin crystallization further progressed by the action of the initial crystals as crystalline nuclei. As a result, the crystallized cordycepin adhered to the undersurface of the mycelial mat (Fig. 2b) and the precipitated cordycepin in the liquid medium (Fig. 2c) were formed. The liquid cyclone had been reported as a method for efficiently isolating and purifying crystals from the culture broth containing both crystals and microbial cells [41]. In the present study, the cordycepin particles like Fig. 2b were difficult to be separated from the mycelia, because stirring at high temperature was not able to completely
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Fig. 2. Photographs of initial cordycepin crystals which can be the crystalline nuclei (a), cordycepin crystals attached to the undersurface of the mycelial mat (b) and precipitated cordycepin (c).
12
100 mL (1.8 cm) 150 mL (2.6 cm) 200 mL (3.5 cm) 250 mL (4.4 cm) 300 mL (5.3 cm)
10
Cordycepin [g/L]
crystallization, the initial medium condition should be changed by other ways.
Medium volume (depth)
8
3.2. Effect of the initial medium volume and the initial substrates concentration on the cordycepin production by G81-3
6 4 2 0 0
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35
40
45
Culture time [d] Fig. 3. Time courses of cordycepin production by G81-3 using the filtered standard medium for different initial volumes (medium depths).
dissolve the particles and overheating might cause extraction of the various impurities from the cells leading to adverse effects on a later purification process. In order to maximize the cordycepin productivity of G81-3, the culture conditions need to be modified. Finding an effective culture condition of G81-3 for a filtered medium without cordycepin crystallization is the main purpose of this study. First of all, the initial medium volume (medium depth) was raised in order to decrease the condensation rate of the liquid medium. Fig. 3 shows the time courses of the cordycepin production for the different initial volumes. Even if the volume was raised to 300 mL, it was impossible to prevent the cordycepin crystallization. To avoid cordycepin
In order to maximize the cordycepin production without its crystallization, the initial medium volume and/or the initial medium concentration were changed. The C/N ratio of the medium remained constant (glucose/yeast extract = 86.2/93.8) in all the experiments and the Medium M consisting of 86.2 g/L glucose and 93.8 g/L yeast extract was defined as the standard. Fig. 4 shows the time courses of the cordycepin production at two levels of substrate concentrations, which were 0.75 (a) and 0.5 (b) to the standard for the different initial medium volume between 100 mL and 300 mL. Only at the combination of 0.75 and 100 mL, the cordycepin was crystallized at a later phase. The final cordycepin production depended on the initial substrate level, whereas it hardly depended on the initial volume. As shown in Fig. 5, the yields of cordycepin per initial yeast extract (the cost of yeast extract is 10 times higher than that of glucose) were almost comparable among all the conditions and the most efficient condition was the combination of 0.75 with 150 mL taking into account the production rate. This condition can provide the simplest way to avoid cordycepin crystallization, however, the saturation point of the cordycepin production by G81-3 at a higher level of substrates cannot be determined by this method. Therefore, the culture with a periodic water supplement was tried to prevent the crystallization at a higher substrate level. Fig. 6 shows a comparison of the time courses between the culture with or without water-supplement. The water evaporation during the culture was 1.18 ± 0.01 mL/d regardless of the initial medium condition. Although the total added water was over the total evaporated water, this addition of water was necessary
12 Medium volume (depth)
(a)
Cordycepin [g/L]
10
(b)
100 mL (1.8 cm) 150 mL (2.6 cm) 200 mL (3.5 cm) 250 mL (4.4 cm) 300 mL (5.3 cm)
8 6 4 2 0 0
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Culture time [d]
35
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45 0
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Culture time [d]
Fig. 4. Time courses of cordycepin production by G81-3 using the filtered medium whose substrate level is 0.75 (a) and 0.5 (b) to the standard for different initial volumes (medium depths).
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Medium depth [cm] 6 0.75 Production rate 0.5 Production rate 0.75 Cordycepin yield 0.5 Cordycepin yield
0.3
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10 8
100 6 Volume (with water-supplement) Volume (without water-supplement) Cor. (with water-supplement) Cor. (without water-supplement) Actual Cor. (without water-supplement)
0 20
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Cordycepin [g/L]
Medium volume [mL]
12 150
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Substrate level
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Culture time [d]
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0 40
Culture time [d] Fig. 6. Comparison of time courses of cordycepin production between watersupplementing and no water-supplementing cultures of G81-3. In the watersupplementing culture, 15 mL of sterilized water was added every 3 days from 15 d of the culture time. Initial medium volume: 150 mL.
Cordycepin production [g/L]
to prevent the crystallization at a later phase. As a result, only the culture without water-supplement caused the cordycepin crystallization. At the end of the culture without water-supplement, all the crystallized particles on the undersurface of the mycelial mat were picked off from the mycelia and mixed into the liquid medium. The whole insoluble particles in the liquid medium were dissolved by stirring on heating and then cordycepin concentration was re-measured after dilution to a measurable concentration. This re-measured concentration means the true value of cordycepin concentration produced by G81-3 throughout the culture without water-supplement (the white square in Fig. 6). The result indicated that the water addition had no effect on the final cordycepin production by G81-3. Moreover, both time courses almost overlapped until the crystallization started. Taking the crystallized cordycepin into account, the production rates of the water-supplementing and no water-supplementing cultures would be the same levels. The crystallization prevents the easy determination of the true cordycepin production, since the values obtained by the measurement of the culture broth show only the dissolved cordycepin concentration. Although the water-supplementing culture requires additional work compared to the no water-supplementing one, it permits an easy evaluation of the upper limit of cordycepin production by G81-3.
5
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0.00 400
Fig. 5. Relation between initial medium volume and cordycepin production rate or cordycepin yield per yeast extract obtained from the results shown in Fig. 4.
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0.4
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Substrate level Fig. 7. Time courses of cordycepin production by G81-3 using the filtered medium (a) and final cordycepin production, cordycepin production rate and cordycepin yield per yeast extract (b) at different substrate levels. Initial medium volume: 150 mL.
3.3. Evaluation of cordycepin production capacity of G81-3 For the autoclaved medium, the cordycepin production reached the maximum in the standard medium defined above [37]. For the filtered medium, the optimum substrate concentration must be different from that of the autoclaved medium. So the effect of the substrate concentrations on the cordycepin production was investigated. Fig. 7a shows the time courses of the cordycepin production at the different substrate concentrations at the fixed C/N ratio (glucose/yeast extract = 86.2/93.8). The substrate level was defined as the ratio of glucose concentration in the medium to that in the standard medium. The initial medium volumes were set at 150 mL in all the cases. When the substrate level was 1.0 or more, the periodic water-supplementing culture was employed. More than 1.0 of the substrate level further increased the cordycepin production and the increase almost stopped at the substrate level 1.6. Based on the results shown in Fig. 7a, the effects of the substrate level on the cordycepin production rate and the yield of cordycepin per initial yeast extract are shown in Fig. 7b. The data indicates two findings. One is that the maximum capacity of the cordycepin production for G81-3 is around 14.3 g/L and the other is that favorable substrate levels to save both time and cost are between 1.0 and 1.4. The highest production rate of 0.36 g/(L d) obtained in this study was slightly lower than the highest value obtained by the repeated batch culture using the autoclaved medium [38]. Application of a repeated batch culture with the medium exchange just before crystallization can eliminate the need for the periodic water-supplement and would easily improve the production rate.
Cordycepin productivity [g-Cor ./(g-DCW .d)]
M. Masuda et al. / Process Biochemistry 49 (2014) 181–187
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(a) Cordycepin [g/L]
10 8 6 4 0.75 Large scale 0.75 Small scale 1.0 Large scale 1.0 Small scale
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Cell surface density (g-DCW/cm )
(b) Cordycepin [g/bottle]
185
Fig. 9. Relation between cell surface density and cordycepin productivity obtained from the results shown in Fig. 4a.
4
3
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0 0
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15
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25
30
35
40
Culture time [d] Fig. 8. Comparison of time courses of cordycepin production [g/L] (a) and net cordycepin production [g/culture bottle] (b) by G81-3 between the small bottle and the large bottle. Substrate level: 0.75 or 1.0; initial medium depth: 2.6 cm.
productivity was observed. This difference might be due to difference in the oxygen uptake rate which is closely related to the oxygen deficiency in the mycelial mat, because the production rate of citric acid by Aspergillus niger (ca 3 g/(L d)) is much higher than that of cordycepin by C. militaris. The efficient supply of the substrates from the liquid medium might be potentially more influential on the cordycepin production than that of oxygen from the upper surface of the mycelia in the liquid surface culture of G81-3. Mao et al. also reported that the DO shift to a lower level during the latter stage of the submerged culture was effective in obtaining a higher cordycepin production [43]. Therefore, the best way of scale-up to have the same efficiency as the small scale might be fixing not the medium depth but other factors like the ratio of the perimeter to depth. This is the subject for a further study.
3.4. Scale-up test for the liquid surface culture of G81-3
3.5. Change in the medium components by autoclave sterilization and their influence on the cell growth of the wild-type strain or the mutant G81-3
A larger culture bottle (the cross-section area is 2.5 times greater than that used in the above-described experiment) was used to check the influences of scale-up on the cordycepin production. According to the paper reported by Sakurai et al., the productivity of citric acid by the surface culture depended on the medium depth [42]. Therefore, in this scale-up test, the medium depth for the large scale was fixed at the same level as that of the small scale. The experiments were carried out in two conditions. One was the substrate level 1.0 with the water-supplement and the other was the substrate level 0.75 without the water-supplement (Fig. 8). In both cases, the final cordycepin productions were hardly affected by the size of the culture bottle, that is, the expansion of the liquid surface area resulted in an improvement of the net production corresponding to the scale-up factor (×2.5). On the other hand, the production rates of both cases were reduced by the expansion of the liquid surface area. Fig. 9 shows the relation between the cell surface density, which is defined as the dry cell weight at the end of the culture divided by the surface area [g-DCW/cm2 ], and the cordycepin productivity [(g-cordycepin)/(g-DCW d)] calculated from the linear part of each time course in Fig. 4a (the cell growth almost reached a maximum at the end of the linear part). Generally, the increase in the cell surface density leads to an oxygen deficiency inside the mycelial mat. In the previous paper about the citric acid production by the surface culture, the productivity drastically decreased with an increase in the cell surface density [42]. As shown in Fig. 9, the quite gradual decrease in the cordycepin
The yeast extract contains abundant amino acids and vitamins, especially vitamin Bs (Difco Manual 11th Edition). During the autoclaving process, the Maillard reaction between amino acids and glucose and other unknown reactions could occur, resulting in the production of unknown compounds. In addition, some heat-labile vitamins, such as vitamin B6 , vitamin B12 , pantothenic acid and folic acid, must be denatured. In Table 1, the actual values of the glucose concentration in the standard media after sterilization are shown. In both media, the autoclave sterilization reduced the free glucose concentration to 82% of that in the corresponding filtered media. The dry cell weight (DCW) at the end of the culture of G81-3 or the wild-type strain using each standard medium (Medium M or Medium W) by autoclaving or filtration are also shown in Table 1. Although the DCW of G81-3 was reduced to about 67% using the autoclaved medium, it was hardly reduced for the wild-type strain. Moreover, the reduction in the DCW significantly influenced the reduction in the cordycepin production. This was consistent with the result that the lag phase was prolonged only in the culture of the mutant with the autoclaved medium as shown in Fig. 1. The primary metabolisms related to the cell growth must be different between the mutant and the wild-type strain. In other words, only for G81-3, some heat-labile vitamins significantly contributed to the growth, and/or certain compounds produced by the autoclave significantly inhibited the growth. The specific cordycepin production [g-cordycepin/g-DCW] was only slightly affected by the sterilization method for both strains as shown in Table 1. This result
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Table 1 Effect of sterilization method on the substrate concentrations in the medium and cordycepin productions. Sterilization
Wild-type (Glc = 62.6 g/La ) Autoclave
Actual glucose (g/L) Actual amino acids (mM) DCW (g/L) Cordycepin (g/L) Cordycepin/DCW (g/g)
48.5 222 29.5 2.4 0.080
± ± ± ± ±
1.7 1 0.2 0.2 0.005
Mutant (Glc = 86.2 g/La ) Filtration 59.2 249 32.6 2.5 0.076
± ± ± ± ±
1.2 3 0.2 0.1 0.003
Autoclave 66.6 293 30.7 6.9 0.227
± ± ± ± ±
0.8 4 3.5 0.3 0.019
Filtration 81.1 331 46.2 10.9 0.237
± ± ± ± ±
2.0 3 2.2 0.4 0.018
Values are mean ± SD of triplicate cultures. a Initially supplied glucose before sterilization.
indicated that the change of the medium components by the autoclave had little effect on the cordycepin biosynthetic process of both strains. Taken together, the reason for the increased cordycepin production of G81-3 by changing sterilization method is the increased cell growth with no change in the cordycepin biosynthetic capacity. Throughout this study, the C/N ratio of the medium was fixed to that of the standard medium previously optimized by the RSM using the autoclaved medium. As shown in Table 1, the reduction ratio of glucose and amino acids by the autoclave was different from each other. This result is assumed to be due to the difference in the molecular weight between the glucose (180) and amino acids (126, the averaged molecular weight of the amino acids in the yeast extract calculated according to the data for the amino acids composition in the Difco & BBL Manual second edition). If the glucose and amino acid reacted at the ratio of 1/1 (mol/mol), the reduction ratio of the amino acids should be lower than that of the glucose. Therefore, the optimum C/N ratio for the filtered medium might slightly shift from that for the autoclaved medium. The future challenge is to adjust the C/N ratio for the filtered medium.
4. Conclusions C. militaris mutant G81-3 was verified to have an enormous capacity for cordycepin production and therefore, the produced cordycepin crystallized during a later culture period. Moreover, a part of the crystallized cordycepin strongly adhered to the mycelia and could not be completely recovered. Finding a more simplified and lower-cost process for cordycepin production by maximize this G81-3 ability is important for its commercial production. In this study, the primary culture conditions, such as medium concentration and medium depth to prevent the cordycepin crystallization, were investigated for the liquid surface culture of G81-3. As a result, two methods were developed: one was that decreasing the initial substrate level and increasing the initial medium volume for the produced cordycepin not to exceed the solubility, and the other was periodic addition of the water in the culture bottle. Furthermore, the scale-up tests of these methods were carried out and the desired net production was achieved.
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Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Efficient production of cordycepin by the Cordyceps militaris mutant G81-3 for practical use Mina Masuda a , Shonkor Kumar Das a , Masanori Hatashita b , Shinya Fujihara a , Akihiko Sakurai a,∗ a b
Department of Applied Chemistry and Biotechnology, Graduate School of Engineering, University of Fukui, 3-9-1 Bunkyo, Fukui 910-8507, Japan Research & Development Department, The Wakasa-wan Research Center, 64-52-1 Nagatani, Tsuruga 914-0192, Japan
a r t i c l e
i n f o
Article history: Received 17 July 2013 Received in revised form 17 October 2013 Accepted 24 October 2013 Available online 1 November 2013 Keywords: Cordycepin Cordyceps militaris mutant Liquid surface culture Scale-up Crystallization
a b s t r a c t Cordycepin is one of the most versatile metabolites of Cordyceps militaris. When C. militaris G81-3, the mutant obtained by a proton beam irradiation, was cultivated by liquid surface culture, cordycepin was found to crystallize in the medium due to high cordycepin concentration. Because the cordycepin crystals strongly attached to the mycelial mat, complete recovery of cordycepin was difficult. To prevent cordycepin crystallization, increase of the initial medium volume was examined to decrease the condensation rate by vaporization, in combination with decrease of the initial substrate level. Besides, addition of the water to the culture bottle was examined to cancel out the medium condensation. A 7.4 g/L of cordycepin was obtained without crystallization by the former method. The upper limit of cordycepin production, 14.3 g/L, was obtained by the latter method and this value was the highest level of all the previous reports. A 2.5-fold scale-up of the culture slightly declined the production rate, however, the amount of cordycepin production was properly 2.5 times higher than that of the small scale. In addition, differences in sensitivities to the substrates between the mutant and the wild-type strain are discussed. © 2013 Elsevier Ltd. All rights reserved.
1. Introduction Medicinal mushrooms have been widely used for the promotion of health and for the prevention and treatment of various diseases due to their biological active constituents [1]. Among them, the fungi of the genus Cordyceps (belonging to Clavicipitaceae, Ascomycetes), which parasitize insects, are considered to be the more promising medicinal mushrooms [2]. Cordyceps sinensis and Cordyceps militaris, two well-known Cordyceps species have multiple physiological and pharmacological activities [3–8] derived from a variety of chemical components such as cordycepin, ergosterol, peptides, polysaccharides, and superoxide dismutase [9–17]. Cordycepin, a nucleoside analog (3 -deoxyadenosine), is the major active constituent of C. militaris and was first isolated from a culture broth of C. militaris in the 1950s [9]. Since then, many biological functions of cordycepin including anti-tumor, anti-metastatic, anti-bacterial, anti-viral, immunomodulatory and anti-inflammatory activities have been discovered [18–26]. In the
∗ Corresponding author. Tel.: +81 776 27 8924; fax: +81 776 27 8747. E-mail address: a [email protected] (A. Sakurai). 1359-5113/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.procbio.2013.10.017
last decade, cordycepin has been studied as a therapeutic agent for a variety of cancers, especially leukemia (ClinicalTrials.gov, Verified by OncoVista, Inc., 2009), trypanosomiasis and restenosis [27–30]. The designs of derivatives were also studied to protect against rapid in vivo oxidation [31]. These studies imply the need for the largescale production of cordycepin. As for the cordycepin production by C. militaris, the liquid surface culture [32], the submerged culture [33], and the combination of submerged and static cultures [34] were investigated to efficiently produce cordycepin. Recently, an efficient method to extract cordycepin from the waste mushroom beds was proposed [35]. In order to improve cordycepin production to a commercial level, a novel mutant was obtained from C. militaris using high-energy proton beam irradiation as reported in our previous study [36]. In the liquid surface culture of this mutant (G81-3), the concentrations of the carbon and nitrogen sources were optimized, and the peak cordycepin production was 2.8 times that of the wild-type strain [37]. In addition, the repeated batch culture method was investigated to achieve a higher production rate [g/(L d)] of cordycepin [38]. In this study, the culture conditions previously reported for G813 [37] was further investigated from the viewpoint of both timeand cost-savings by the liquid surface culture. The ultimate purpose of this research is the abundant supply of cordycepin in order to discover a novel pharmacological use and to advance the clinical trials of this compound.
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2. Materials and methods 2.1. Microorganisms and culture
2.2. Analytical methods The concentration of cordycepin was determined by an HPLC equipped with a UV detector (LC-9A system, Shimadzu Corp., Japan) and a reverse phase column (TSK-gel ODS-80Ts, Tosoh Corp., Japan). The mobile phase consisted of methanol and 0.1% (v/v) phosphoric acid (2/98, v/v). The flow rate was 1.0 mL/min, and the column temperature was 40 ◦ C. The chromatogram was monitored by the UV absorbance at 260 nm. The authentic standard was purchased from Sigma–Aldrich Co. The glucose concentration was measured by the mutarotase-GOD (glucose oxidase) method using a Glucose CII Test Wako (Wako Pure Chemical Industries, Ltd., Japan). The amino acid concentration was determined by measuring the absorbance at 420 nm of the reaction product of the primary amino groups with sodium 2, 4, 6-trinitrobenzenesulfonate (TNBS·Na) in the presence of sodium sulfite at pH 9.9 [40]. Since the sensitivity of proteins was much lower than that of the free amino acids, the detected value could be considered as that derived from the free amino acids. l-glycine was used for preparing the calibration curve. To determine the dry cell weight (DCW), the entire contents of the culture bottle were centrifuged (20 min at 15,000 × g). The separated mycelia were sufficiently washed with distilled water and freeze-dried for 48 h, then, the weight was measured. To measure the content of cordycepin in the crystallized compounds during the culture, all the insoluble particles, including the particles precipitated in the liquid medium and the particles attached to the mycelial mat, were collected and dried out on the filter paper. The attached particles were collected one by one with the tweezers. They were dissolved in the heated water (40 ◦ C) and the suspended solid were removed by filtration, then the filtrate was freeze-dried. After measurement of weight of all the freeze-dried products, it was dissolved in distilled water at 100 mg/L and the cordycepin concentration was determined by the HPLC. The cordycepin content in the crystallized compounds was calculated based on the cordycepin concentration of the said solution. To determine the volume of the evaporated water from the liquid medium, total weight of the culture bottle was measured periodically and the decrease in the weight was regarded as the volume of the water evaporation. All of the cordycepin concentrations shown in this experiment were corrected values considering the condensation or dilution of the medium due to vaporization or the addition of water. These values were obtained by dividing the total produced cordycepin (g) by the initial medium volume (L).
Cordycepin [g/L]
C. militaris NBRC 9787 and the mutant G81-3 [36] were used in the present experiments. The wild-type strain was stored on a potato dextrose agar (PDA) (Nissui Pharmaceutical Co., Ltd., Japan) slant, and the mutant was on an agar slant of Vogel’s medium [39] supplemented with 200 mM of 8-azaguanine at 5 ◦ C. Prior to the experiment, a piece of mycelia in the stock culture was transferred to a fresh PDA slant and incubated at 25 ◦ C for eight days. The seed culture was started by transferring one loopful of mycelia from each active slant to a PDA plate and incubated at 25 ◦ C for 13 days for the wild-type strain and for 20 days for the mutant. The inoculum was prepared by punching out a 1-cm disk from the PDA plate with a sterilized cylindrical cutter. The liquid surface culture was started by inoculating two seed disks into a 500-mL culture bottle, or five seed disks into a 1250-mL culture bottle. The diameter of the former bottle was 8.5 cm and that of the latter one was 13.5 cm. The heights of both were 14.0 cm with bottlenecks of 4.5 cm diameter and 4.0 cm height. The initial volume of the liquid medium was set between 100 mL and 300 mL (corresponding to 1.8 cm and 5.3 cm of medium depth) for the small bottle and was 378 mL (corresponding to 2.6 cm of medium depth) for the large one. The medium volume decreased during the culture by water evaporation from the liquid surface. In contrast, it increased when sterile distilled water was supplemented to avoid the medium condensation. In the latter case, a supporter (a stainless-steel round frame) was hung in the bottle in order to prevent the mycelial mat from soaking in the liquid medium. The bottleneck was filled with a cotton plug during the culture. The prepared bottles were placed in an incubator maintained at a temperature of 25 ± 1 ◦ C and a humidity of 30 ± 2%. Just before sampling, the medium was gently agitated for 5 s by a magnetic stirrer. A 1-mL portion of the medium was withdrawn through a sampling port typically at a 3-day interval and was filtered through a 0.45-m membrane filter in order to remove the suspended mycelia. The filtrate was analyzed for cordycepin, glucose and pH. The standard media for the wild-type strain and the mutant are as follows. For the wild-type strain, the medium (Medium W) was composed of 62.6 g/L of glucose and 72.5 g/L of yeast extract (Difco); for the mutant, the medium (Medium M) was composed of 86.2 g/L of glucose and 93.8 g/L of yeast extract. Both media contained Vogel’s medium diluted to a 1/10 concentration. These standard media had been determined in a previous investigation using the response surface method (RSM) to obtain the optimum concentration of the carbon and nitrogen sources for the cordycepin production. To check the reproducibility, the experiments were carried out at least triplicate.
Mutant Autoclave Mutant Filtration Wild-type Autoclave Wild-type Filtration
10 8 6 4 2 0 0
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Cultrure time [d] Fig. 1. Comparison of time courses of cordycepin production by the wild-type strain and G81-3 using the filtered medium and the autoclaved medium. Initial medium volume: 100 mL.
3. Results and discussion 3.1. Effects of the medium sterilization process on cordycepin production by C. militaris or its mutant In the previous investigation using the RSM, the optimum medium concentration was extremely high. Nevertheless, the media had been sterilized by autoclave in the overall experiments. Therefore, the deactivation of vitamins and synthesis of unknown compounds by the Maillard reaction during autoclave might have some adverse effects on the cordycepin production. In order to determine the effect of the autoclave sterilization, the cordycepin production using the autoclaved medium was compared to that using the filtered one. Fig. 1 shows the time courses of the cordycepin production using the autoclaved medium and the filtered medium. (In all the figures, the symbols show the average values and the error bars show the minimum and maximum values.) Regardless of the sterilization method, the culture periods and final cordycepin productions were almost the same for the wild-type strain. On the other hand, for the mutant G81-3, the time courses showed significant differences between the sterilization methods. The use of the filtered medium significantly reduced the lag phase. The prolonged lag phase with the autoclaved medium was considered to be due to some inhibiting effect of unknown compounds synthesized by the autoclave on the growth of G81-3, since the cell growth and the glucose consumption were hardly observed before 15 d of culture time. In addition, the filtered medium increased cordycepin production and caused crystallization after 24 d. The content of cordycepin in the crystallized compounds was over 97%, and therefore the dissolved cordycepin concentration decreased due to the transfer of cordycepin from the liquid medium to the crystallized compounds. The produced cordycepin in the medium had kept oversaturation for a while (the solubility of cordycepin in water is approximately 7 g/L at 25 ◦ C), however, the locally-concentrated cordycepin around the mycelial mat started to crystallize as shown in the white circle of Fig. 2a. Cordycepin crystallization further progressed by the action of the initial crystals as crystalline nuclei. As a result, the crystallized cordycepin adhered to the undersurface of the mycelial mat (Fig. 2b) and the precipitated cordycepin in the liquid medium (Fig. 2c) were formed. The liquid cyclone had been reported as a method for efficiently isolating and purifying crystals from the culture broth containing both crystals and microbial cells [41]. In the present study, the cordycepin particles like Fig. 2b were difficult to be separated from the mycelia, because stirring at high temperature was not able to completely
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Fig. 2. Photographs of initial cordycepin crystals which can be the crystalline nuclei (a), cordycepin crystals attached to the undersurface of the mycelial mat (b) and precipitated cordycepin (c).
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100 mL (1.8 cm) 150 mL (2.6 cm) 200 mL (3.5 cm) 250 mL (4.4 cm) 300 mL (5.3 cm)
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Cordycepin [g/L]
crystallization, the initial medium condition should be changed by other ways.
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3.2. Effect of the initial medium volume and the initial substrates concentration on the cordycepin production by G81-3
6 4 2 0 0
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Culture time [d] Fig. 3. Time courses of cordycepin production by G81-3 using the filtered standard medium for different initial volumes (medium depths).
dissolve the particles and overheating might cause extraction of the various impurities from the cells leading to adverse effects on a later purification process. In order to maximize the cordycepin productivity of G81-3, the culture conditions need to be modified. Finding an effective culture condition of G81-3 for a filtered medium without cordycepin crystallization is the main purpose of this study. First of all, the initial medium volume (medium depth) was raised in order to decrease the condensation rate of the liquid medium. Fig. 3 shows the time courses of the cordycepin production for the different initial volumes. Even if the volume was raised to 300 mL, it was impossible to prevent the cordycepin crystallization. To avoid cordycepin
In order to maximize the cordycepin production without its crystallization, the initial medium volume and/or the initial medium concentration were changed. The C/N ratio of the medium remained constant (glucose/yeast extract = 86.2/93.8) in all the experiments and the Medium M consisting of 86.2 g/L glucose and 93.8 g/L yeast extract was defined as the standard. Fig. 4 shows the time courses of the cordycepin production at two levels of substrate concentrations, which were 0.75 (a) and 0.5 (b) to the standard for the different initial medium volume between 100 mL and 300 mL. Only at the combination of 0.75 and 100 mL, the cordycepin was crystallized at a later phase. The final cordycepin production depended on the initial substrate level, whereas it hardly depended on the initial volume. As shown in Fig. 5, the yields of cordycepin per initial yeast extract (the cost of yeast extract is 10 times higher than that of glucose) were almost comparable among all the conditions and the most efficient condition was the combination of 0.75 with 150 mL taking into account the production rate. This condition can provide the simplest way to avoid cordycepin crystallization, however, the saturation point of the cordycepin production by G81-3 at a higher level of substrates cannot be determined by this method. Therefore, the culture with a periodic water supplement was tried to prevent the crystallization at a higher substrate level. Fig. 6 shows a comparison of the time courses between the culture with or without water-supplement. The water evaporation during the culture was 1.18 ± 0.01 mL/d regardless of the initial medium condition. Although the total added water was over the total evaporated water, this addition of water was necessary
12 Medium volume (depth)
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Fig. 4. Time courses of cordycepin production by G81-3 using the filtered medium whose substrate level is 0.75 (a) and 0.5 (b) to the standard for different initial volumes (medium depths).
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Medium depth [cm] 6 0.75 Production rate 0.5 Production rate 0.75 Cordycepin yield 0.5 Cordycepin yield
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Culture time [d] Fig. 6. Comparison of time courses of cordycepin production between watersupplementing and no water-supplementing cultures of G81-3. In the watersupplementing culture, 15 mL of sterilized water was added every 3 days from 15 d of the culture time. Initial medium volume: 150 mL.
Cordycepin production [g/L]
to prevent the crystallization at a later phase. As a result, only the culture without water-supplement caused the cordycepin crystallization. At the end of the culture without water-supplement, all the crystallized particles on the undersurface of the mycelial mat were picked off from the mycelia and mixed into the liquid medium. The whole insoluble particles in the liquid medium were dissolved by stirring on heating and then cordycepin concentration was re-measured after dilution to a measurable concentration. This re-measured concentration means the true value of cordycepin concentration produced by G81-3 throughout the culture without water-supplement (the white square in Fig. 6). The result indicated that the water addition had no effect on the final cordycepin production by G81-3. Moreover, both time courses almost overlapped until the crystallization started. Taking the crystallized cordycepin into account, the production rates of the water-supplementing and no water-supplementing cultures would be the same levels. The crystallization prevents the easy determination of the true cordycepin production, since the values obtained by the measurement of the culture broth show only the dissolved cordycepin concentration. Although the water-supplementing culture requires additional work compared to the no water-supplementing one, it permits an easy evaluation of the upper limit of cordycepin production by G81-3.
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Fig. 5. Relation between initial medium volume and cordycepin production rate or cordycepin yield per yeast extract obtained from the results shown in Fig. 4.
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0.00 2.0
Substrate level Fig. 7. Time courses of cordycepin production by G81-3 using the filtered medium (a) and final cordycepin production, cordycepin production rate and cordycepin yield per yeast extract (b) at different substrate levels. Initial medium volume: 150 mL.
3.3. Evaluation of cordycepin production capacity of G81-3 For the autoclaved medium, the cordycepin production reached the maximum in the standard medium defined above [37]. For the filtered medium, the optimum substrate concentration must be different from that of the autoclaved medium. So the effect of the substrate concentrations on the cordycepin production was investigated. Fig. 7a shows the time courses of the cordycepin production at the different substrate concentrations at the fixed C/N ratio (glucose/yeast extract = 86.2/93.8). The substrate level was defined as the ratio of glucose concentration in the medium to that in the standard medium. The initial medium volumes were set at 150 mL in all the cases. When the substrate level was 1.0 or more, the periodic water-supplementing culture was employed. More than 1.0 of the substrate level further increased the cordycepin production and the increase almost stopped at the substrate level 1.6. Based on the results shown in Fig. 7a, the effects of the substrate level on the cordycepin production rate and the yield of cordycepin per initial yeast extract are shown in Fig. 7b. The data indicates two findings. One is that the maximum capacity of the cordycepin production for G81-3 is around 14.3 g/L and the other is that favorable substrate levels to save both time and cost are between 1.0 and 1.4. The highest production rate of 0.36 g/(L d) obtained in this study was slightly lower than the highest value obtained by the repeated batch culture using the autoclaved medium [38]. Application of a repeated batch culture with the medium exchange just before crystallization can eliminate the need for the periodic water-supplement and would easily improve the production rate.
Cordycepin productivity [g-Cor ./(g-DCW .d)]
M. Masuda et al. / Process Biochemistry 49 (2014) 181–187
12
(a) Cordycepin [g/L]
10 8 6 4 0.75 Large scale 0.75 Small scale 1.0 Large scale 1.0 Small scale
2 0 5
0.025
0.020
0.015
0.010
0.005
0.000 0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
2
Cell surface density (g-DCW/cm )
(b) Cordycepin [g/bottle]
185
Fig. 9. Relation between cell surface density and cordycepin productivity obtained from the results shown in Fig. 4a.
4
3
2
1
0 0
5
10
15
20
25
30
35
40
Culture time [d] Fig. 8. Comparison of time courses of cordycepin production [g/L] (a) and net cordycepin production [g/culture bottle] (b) by G81-3 between the small bottle and the large bottle. Substrate level: 0.75 or 1.0; initial medium depth: 2.6 cm.
productivity was observed. This difference might be due to difference in the oxygen uptake rate which is closely related to the oxygen deficiency in the mycelial mat, because the production rate of citric acid by Aspergillus niger (ca 3 g/(L d)) is much higher than that of cordycepin by C. militaris. The efficient supply of the substrates from the liquid medium might be potentially more influential on the cordycepin production than that of oxygen from the upper surface of the mycelia in the liquid surface culture of G81-3. Mao et al. also reported that the DO shift to a lower level during the latter stage of the submerged culture was effective in obtaining a higher cordycepin production [43]. Therefore, the best way of scale-up to have the same efficiency as the small scale might be fixing not the medium depth but other factors like the ratio of the perimeter to depth. This is the subject for a further study.
3.4. Scale-up test for the liquid surface culture of G81-3
3.5. Change in the medium components by autoclave sterilization and their influence on the cell growth of the wild-type strain or the mutant G81-3
A larger culture bottle (the cross-section area is 2.5 times greater than that used in the above-described experiment) was used to check the influences of scale-up on the cordycepin production. According to the paper reported by Sakurai et al., the productivity of citric acid by the surface culture depended on the medium depth [42]. Therefore, in this scale-up test, the medium depth for the large scale was fixed at the same level as that of the small scale. The experiments were carried out in two conditions. One was the substrate level 1.0 with the water-supplement and the other was the substrate level 0.75 without the water-supplement (Fig. 8). In both cases, the final cordycepin productions were hardly affected by the size of the culture bottle, that is, the expansion of the liquid surface area resulted in an improvement of the net production corresponding to the scale-up factor (×2.5). On the other hand, the production rates of both cases were reduced by the expansion of the liquid surface area. Fig. 9 shows the relation between the cell surface density, which is defined as the dry cell weight at the end of the culture divided by the surface area [g-DCW/cm2 ], and the cordycepin productivity [(g-cordycepin)/(g-DCW d)] calculated from the linear part of each time course in Fig. 4a (the cell growth almost reached a maximum at the end of the linear part). Generally, the increase in the cell surface density leads to an oxygen deficiency inside the mycelial mat. In the previous paper about the citric acid production by the surface culture, the productivity drastically decreased with an increase in the cell surface density [42]. As shown in Fig. 9, the quite gradual decrease in the cordycepin
The yeast extract contains abundant amino acids and vitamins, especially vitamin Bs (Difco Manual 11th Edition). During the autoclaving process, the Maillard reaction between amino acids and glucose and other unknown reactions could occur, resulting in the production of unknown compounds. In addition, some heat-labile vitamins, such as vitamin B6 , vitamin B12 , pantothenic acid and folic acid, must be denatured. In Table 1, the actual values of the glucose concentration in the standard media after sterilization are shown. In both media, the autoclave sterilization reduced the free glucose concentration to 82% of that in the corresponding filtered media. The dry cell weight (DCW) at the end of the culture of G81-3 or the wild-type strain using each standard medium (Medium M or Medium W) by autoclaving or filtration are also shown in Table 1. Although the DCW of G81-3 was reduced to about 67% using the autoclaved medium, it was hardly reduced for the wild-type strain. Moreover, the reduction in the DCW significantly influenced the reduction in the cordycepin production. This was consistent with the result that the lag phase was prolonged only in the culture of the mutant with the autoclaved medium as shown in Fig. 1. The primary metabolisms related to the cell growth must be different between the mutant and the wild-type strain. In other words, only for G81-3, some heat-labile vitamins significantly contributed to the growth, and/or certain compounds produced by the autoclave significantly inhibited the growth. The specific cordycepin production [g-cordycepin/g-DCW] was only slightly affected by the sterilization method for both strains as shown in Table 1. This result
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Table 1 Effect of sterilization method on the substrate concentrations in the medium and cordycepin productions. Sterilization
Wild-type (Glc = 62.6 g/La ) Autoclave
Actual glucose (g/L) Actual amino acids (mM) DCW (g/L) Cordycepin (g/L) Cordycepin/DCW (g/g)
48.5 222 29.5 2.4 0.080
± ± ± ± ±
1.7 1 0.2 0.2 0.005
Mutant (Glc = 86.2 g/La ) Filtration 59.2 249 32.6 2.5 0.076
± ± ± ± ±
1.2 3 0.2 0.1 0.003
Autoclave 66.6 293 30.7 6.9 0.227
± ± ± ± ±
0.8 4 3.5 0.3 0.019
Filtration 81.1 331 46.2 10.9 0.237
± ± ± ± ±
2.0 3 2.2 0.4 0.018
Values are mean ± SD of triplicate cultures. a Initially supplied glucose before sterilization.
indicated that the change of the medium components by the autoclave had little effect on the cordycepin biosynthetic process of both strains. Taken together, the reason for the increased cordycepin production of G81-3 by changing sterilization method is the increased cell growth with no change in the cordycepin biosynthetic capacity. Throughout this study, the C/N ratio of the medium was fixed to that of the standard medium previously optimized by the RSM using the autoclaved medium. As shown in Table 1, the reduction ratio of glucose and amino acids by the autoclave was different from each other. This result is assumed to be due to the difference in the molecular weight between the glucose (180) and amino acids (126, the averaged molecular weight of the amino acids in the yeast extract calculated according to the data for the amino acids composition in the Difco & BBL Manual second edition). If the glucose and amino acid reacted at the ratio of 1/1 (mol/mol), the reduction ratio of the amino acids should be lower than that of the glucose. Therefore, the optimum C/N ratio for the filtered medium might slightly shift from that for the autoclaved medium. The future challenge is to adjust the C/N ratio for the filtered medium.
4. Conclusions C. militaris mutant G81-3 was verified to have an enormous capacity for cordycepin production and therefore, the produced cordycepin crystallized during a later culture period. Moreover, a part of the crystallized cordycepin strongly adhered to the mycelia and could not be completely recovered. Finding a more simplified and lower-cost process for cordycepin production by maximize this G81-3 ability is important for its commercial production. In this study, the primary culture conditions, such as medium concentration and medium depth to prevent the cordycepin crystallization, were investigated for the liquid surface culture of G81-3. As a result, two methods were developed: one was that decreasing the initial substrate level and increasing the initial medium volume for the produced cordycepin not to exceed the solubility, and the other was periodic addition of the water in the culture bottle. Furthermore, the scale-up tests of these methods were carried out and the desired net production was achieved.
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