Utilization of Corncob Biochar in Cultivation Media for Cordycepin Production and Biomass of Cordyceps militaris

Abstract

Cordyceps militaris is an entomopathogenic fungus. It is well-known as a rich source of bioactive compounds called cordycepins and adenosines, which are useful in medicinal applications. The effects of medium components on cordycepin and adenosine production by C. militaris, obtained by adding different conditions of corncob biochar in solid media, were investigated in this study. The medium components, which mixed 0.1, 0.3, 0.5, 1, 5 and 10 g of biochar with rice berries, were optimized to improve the yield of biomasses, cordycepins, and adenosines. The results showed that 10 g of biochar mixed with a rice berry medium was the optimal medium condition for the highest dry fruiting body weight (DFW) and cordycepin yield (CY) at 3.6 kg/bottle and 20.5 mg/g, respectively, but the adenosine yield (AY) was similar to that in other conditions. Moreover, the SEM showed that the mycelia of C. militaris attached to the biochar surface (pores) and used it as the resident. EDS analysis from the basal medium indicated that C and O were present in the mycelia of C. militaris with the average values of 25.6% and 71.4%, respectively. This study provides an effective cultivation method by using agricultural residue, and biochar corncob as a high concentration of carbon for increasing the biomass, cordycepin, and adenosine yield of C. militaris. The information obtained in this study is fundamental and useful to the development of a C. militaris cultivation process for the efficient production of cordycepin on a large scale. The findings suggest that the system design of the cultivation medium is crucial for growth and cordycepin production.

1. Introduction

Cordyceps is the largest and the most diverse genus classified in the family Clavicipitacease, in regard to the number of its species, its morphology, and the variation in its host, with over 750 identified species [1,2]. The species Cordyceps militaris has been widely regarded as a substitute for Ophiocordyceps sinensis, because C. militaris contains similar secondary metabolites in its stromata (e.g., cordycepin) as O. sinensis. It has a worldwide distribution and is easily cultured [3]. Many species of Cordyceps have been used in health foods and medicines in China and South-East Asia as it contains abundant physiologically active substances, such as antagonism by theophylline, potentiation by dipyridamole, and the inhibition of ADP-induced platelet aggregation [4]. Due to recent advancements in pharmaceutical biotechniques, it is possible to isolate bioactive compounds from Cordyceps and make them available in powder and capsular form. Currently, both C. militaris and O. sinensis are used in functional foods and medicines in Asia [5,6]. Many studies have been conducted on the culture requirements for the metabolite production of filamentous fungi [7,8,9,10]. Similarly, the in vitro mycelium growth and fruiting body formation of C. militaris have attracted mycologists, entomologists, and biotechnologists [11]. There have been many studies on the medium composition and culture conditions [12,13,14] for the yield of mycelia and cordycepin production by liquid fermentation in C. militaris. Moreover, some of these studies investigated the culture condition and medium components for the production of mycelial biomasses and exo-polysaccharides with C. militaris in liquid cultures [14]. The concentrations of the mycelial biomasses, exo-polysaccharides, and cordycepin achieved in liquid cultures were quite different between C. militaris and O. sinensis [15]. Therefore, using C. militaris as a substitute for its famous cousin is highly questionable. C. militaris naturally has a slow growth in lepidopteran larvae. Hence, artificial cultures are a more favourable solution for the biotechnological production of C. militaris for a high cordycepin yield (CY). Previously, raw materials, such as rice [16,17], yeast extract [18], and peptone [19] were used as the main components in the cultivation media of C. militaris, but the growth and cordycepin production were still low [20]. Because the cultivation processes are complicated, and the natural fruiting bodies of Cordyceps are very rare and costly to collect, fruiting body production in vitro is not repeatable, and the cordycepin content of natural Cordyceps is much lower than that of cultured mycelia [21].

There have been many studies on the medium composition and culture conditions, such as: the effect of oxygen supply [10], the influence of the initial pH value, nitrogen sources, plant oil and modes of propagation [12], using additives for the optimal culture condition [13], the effect of ion-beam irradiation [18], the effect of pH, temperature and incubation time [22], and using casein hydrolysate in submerged conditions [23] for the yield of mycelia and cordycepin production by liquid fermentation in C. militaris. It is well-known that the growth rate and metabolite production of fungus is affected by many conditions, such as media components, temperature, light, pH, and humidity. However, one the major factors is the media component. There have been some studies on the effects of the medium components on cordycepin production by using various carbon and nitrogen sources. The researchers found that glucose and casein hydrolysate (CH) were most effective as carbon and nitrogen sources in cordycepin production (with a 2.3-fold improvement), respectively, with maximal cordycepin production [24]. Another study investigated cultivated fruiting body production using different growth chambers. Cultivated C. militaris was present in three different chambers: the commercial growth chamber, modified beverage cooler chamber, and culture room during the first week (in dark conditions). The growth rate of mycelia cultivated in the culture room was significantly the highest and fastest on the fruiting body, followed by that of the modified beverage cooler chamber. In the second week (in light conditions), the aerial mycelia that was incubated in the commercial growth chamber and modified beverage cooler chamber produced yellow pigment, but the aerial mycelia in the culture room did not develop pigment. This indicated that light intensity may significantly affect the diverse development of mycelial pigmentation [25].

In this study, the utilization of biochar and agricultural residues for cultivation media were applied. Biochar is a type of black carbon produced from a carbonaceous material through the application of heat or chemicals [26]. Biochar is a stable substrate created from organic material that has been combusted under low- or no-oxygen conditions through the process of pyrolysis [27]. Biochar may increase soil pH, nutrient retention, cation exchange capacity (CEC), crop biomass, and many other variables important to soil quality and agriculture, including increased soil sequestration [27]. Therefore, the carbon source is a major component of the cultivation medium and plays an important role in the growth and production of bioactive compounds by basidiomycetes and other fungi. Various compounds can be used as a carbon and energy source by fungi. Carbon compounds range from simple, small molecules, such as sugars, organic acids, and alcohols, to proteins, polysaccharides, and lipids [28]. Thus, using biochar, which contains small molecules of carbon sources for Cordyceps growing, can enhance the dry fruiting body weight (DFW), as well as the cordycepin and adenosine yields (AY). Therefore, our study aimed to optimize the conditions for culturing C. militaris and to establish bioactive compounds produced by C. militaris cultivated from biochar.

2. Materials and Methods

2.1. Preparation of Cordyceps militaris Mycelium

The C. militaris strain, P-1-012, was obtained from the Engineering Research Center of Southwest Bio-Pharmaceutical Resources, Ministry of Education, Guizhou University, China. The C. militaris (P-1-012) was cultivated on potato dextrose agar (PDA), incubating for 7–10 days. Then, the mycelium was transferred to a 500 mL flask containing 100 mL of potato dextrose broth (PDB) and incubated in an incubator shaker at 18 °C and 80% humidity with 154 rpm for 7 days. A small piece of the seed culture was grown in a 250 mL flask of PDB and inoculated with 50 mL of seed culture to a solid medium which was mixed with biochar at different concentrations. The corncob biochar was synthesized with an in-house modification pyrolysis process [29]. The chemical composition of the biochar is listed in

Table 1. Characteristics of corncob biochar after pyrolysis at 500 °C [29].

Chemical Compositions	Unit	Biochar
C	%wt	80.60 ± 0.38
H	%wt	2.08 ± 0.01
N	%wt	0.58 ± 0.01
S	%wt	0.01 ± 0.00
O	%wt	6.72 ± 0.27
Volatile	%wt	10.94 ± 0.41
Fixed carbon	%wt	76.43 ± 0.43
Ash	%wt	5.02 ± 0.07
TOC	g kg−1	700.06

.

2.2. Cultivation of Cordyceps militaris in Solid Media

The solid media contained 15 g of rice berry mixed with biochar in six conditions: 0.1, 0.3, 0.5, 1, 5, and 10 g; the control was prepared without adding biochar. Each condition contained ten replications. The cultivars were incubated at 20 °C for 40 days. Growth characteristics in batch culture height of each fruiting body were measured, and each treatment’s average was calculated. The fresh weight from each treatment was measured after being harvested. Dried weight was measured after incubating at 30 °C for two days.

2.3. Cordycepin and Adenosine Analyses Using High-Performance Liquid Chromatography (HPLC)

Dried fruiting body of C. militaris powder (0.5 g) was extracted with 20 mL by microwave extraction at 1000 watts for 80 min. The extracts were cooled at room temperature and then filtrated with a Büchner funnel with a No.04 Watchman filter. The filtrations were centrifuged by a centrifuge machine at 9000 rpm for 20 min. The supernatant was lyophilized at −40 to −60 °C for 24 h. Then, we dissolved the dried samples with water (HPLC grade) and filtrated them with a microporous membrane (0.22 µm). The samples were analyzed with HPLC; the elution was performed at a flow rate of 0.5 mL/min and a UV wavelength of 260 nm [30]. Standards of cordycepin and adenosine were purchased from PhytoLab GmbH & Co. (Vestenbergsgreuth, Germany) and Sigma Chemical Corporation (St. Louis, MO, USA), respectively. The linear regression formulae obtained were as follows: cordycepin: y = 10.835x + 2.2564 (R² = 0.9999), and adenosine: y = 61.859x + 11.674 (R² = 1).

2.4. Mycelium Characteristics of C. militaris in Solid Media

The samples of solid media with C. militaris mycelia were observed. The attachment of the biochar surface with the mycelium of C. militaris was characterized using scanning electron microscopy (SEM, TESCAN (MIRA4), 2.5 nm, BSE) at 5 kV. The sample for SEM analysis was coated with a gold stub attached to carbon tape and mounted into a sample holder for analysis. Energy-dispersive X-ray spectroscopy (EDS) allowed for targeted analysis of the sample surfaces (40 mm² Oxford Ultim Max EDS) in order to determine the contents of C and O in the mycelium of C. militaris.

3. Results

3.1. Biomass of C. militaris in Different Media Components

The fruiting body of C. militaris from various solid cultivation media (rice berries with 0.1, 0.3, 0.5, 1, 5, and 10 g of biochar) were harvested after 50 days (Figure 1). As shown in Figure 2, the wet and dry weights and percentages continuously fluctuated between the different conditions. The highest dry fruiting body weight (DFW) was from the rice berry with 10 g of biochar with a 3.685 g DFW. Biochar as a carbon source was investigated by mixing the different amounts of biochar in the culture. Therefore, carbon may play a key role in increasing the fruiting body. Masuda et al. (2006) [18] demonstrated that cordycepin synthesized by C. militaris was present in the culture medium, which was a mixture of pepton and yeast extract, in which the yeast extraction ratio was greater than 75%. The optimal nitrogen ratio (w/w), when glucose and the mixture of peptone and yeast extraction (P/Y = 1/3) were used as the carbon and nitrogen sources, respectively, was 2/1 [18]. The result in this study was considerably lower compared with that in previous studies, such as the study performed by Sung et al. (2010) [5], who presented the yield of the fruiting body weight as 8.14 g during 50 days, and Kim et al. (2010) [31] who obtained a high yield of 12.9 g during 60 days. However, our paper reports the CY and AY contents, which were presented neither in the papers of Kim et al. (2010) [31] nor Lin et al. (2010) [32].

3.2. Cordycepin and Adenosine Production

The optimization of the biochar concentrations in the media conditions were studied. This study investigated the differences of fruiting body weight and the cordycepin production of fruiting bodies when biochar was added. Biochar has the characteristics of high surface area and porosity, which may influence pore and mass transfer in solid media; therefore, biochar was adopted in the solid medium for cordycepin production in this study. Biochar derived from corncobs was added to the medium for C. militaris cultivation, and the statistical results are shown in Figure 3. As shown in Figure 3, all cultures with the biochar addition produced higher cordycepin yields than those in the control medium. A maximum cumulative cordycepin yield of 20.05 mg/g was obtained, whereas the cordycepin production in the non-biochar medium (control) was around 8 mg/g. Therefore, the adenosine content in the fruiting bodies was small, and only achieved a mass between 0.43 and 0.55 g/kg during 50 days from six different condition concentrations of biochar.

The addition of biochar to solid media increased the cordycepin production in response to the cordycepin harvest from C. militaris, and the adenosine yield did not vary from other conditions (

Table 2. Cordycepin and adenosine yields in different solid media conditions.

Condition	Cordycepin Content (mg/g)	Adenosine Content (mg/g)
Rice berry + 0.1 g biochar	11.73	0.55
Rice berry + 0.3 g biochar	11.08	0.49
Rice berry + 0.5 g biochar	12.4	0.43
Rice berry + 1 g biochar	14.19	0.45
Rice berry + 5 g biochar	14.24	0.43
Rice berry + 10 g biochar	20.05	0.44
Control	10.17	0.45

). In this study, biochar proved to significantly enhance the content of cordycepin in C. militaris fruiting bodies. The biochar addition not only increased the surface area available for cell immobilization and microbial expansion, but it also led to an increased oxygen supply and a stable environment in solid media for cell metabolism [6]. As a result, the cordycepin production increased.

3.3. SEM Results

The images of the C. militaris mycelia in basal solid media were illustrated with SEM and shown in Figure 4. The mycelium of C. militaris cultivated in rice berries and mixed with 10 g of corncob biochar, which showed the highest cordycepin content, was selected for this experiment. The biochar images exhibited a rough surface morphology and porosity with various sizes of small canals in the biochar material (Figure 4a,c,d). As shown in Figure 4a–d, the mycelia of C. militaris grew and tightened to different materials, including rice berries and biochar. The mass percentages of the elements were recorded at three points, according to the results of the EDS spectra. The EDS analysis indicated that C and O were present in the mycelia of C. militaris with average values of 25.6% and 71.4%, respectively. Therefore, with an increase in biochar content, which presented high percentages of C, O, and TOC contents as shown in Table 1, the cordycepin content increased.

4. Discussion

Metabolite synthesis by microorganisms is a dynamic process that alters according to the cultivation time and culture conditions [5]. One of the most important biologically active metabolites isolated from C. militaris is cordycepin (3’-deoxyadenosine). Most cordycepins were synthesized by C. militaris in the culture media in previous studies [18], and were low in the fruiting bodies. The results in several investigations were not consistent, according to

Table 3. Weight of the fruiting bodies, generation of cordycepin in the fruiting bodies, and medium by solid-state fermentation in various studies.

No.	Strain	Fruit Bodies Weight (g)	Cordycepin in Fruit Bodies (mg/g)	Cordycepin in Medium (mg/g)	Cultivation Time (days)	Reference
1	C. militaris	8.14			50	[1]
2	C. militaris CM-2			5.49	35	[5]
3	C. militaris CM016			18.92	39	[5]
4	C. militaris		9.7			[35]
5	C. militaris C-10376	12.9			60	[31]
6	C. militaris 067	1.35	18.7	2.1	70	[33]
7	C. militaris		4.6		57–60	[32]
8	C. militaris		5.57		50	[31]
9	C. militaris JF-1			6.0	13	[36]
10	C. militaris cmily-02			1.85	16	[37]
11	C. militaris	52.8	0.082	0.135	21	[38]
12	C. militaris	2.11	15.63	23.17	33	[39]
13	C. militaris P-1-012	3.685	20.05		50	In this study

. The highest report for fruiting body weight was 12.9 g/bottle at 60 days [30], and the maximum cordycepin production in fruiting bodies, except our study, was 18.7 mg/g at 70 days [33], in which the incubation time took longer than in our study [32]. However, in this study, the cordycepin production of C. militaris was studied with fruiting bodies under cereal grain solid media with various conditions of biochar, because component media, such as carbon and nitrogen sources, are effective in cordycepin production. The results showed that the highest cordycepin production of 20.09 mg/g occurred in 50 days (Table 3).

As a result, increasing the mass of biochar in the media increased the yield of cordycepin production (Figure 3). Therefore, biochar was shown to be an effective additive. The pores in corncob biochar related with temperature to produce pores [34]. As shown in the SEM images (Figure 4a–d) of the C. militaris mycelia in basal solid media, the mycelia of C. militaris cultivated in corncob biochar were porous. With their rough surface morphology and various-size porosity in the biochar material, the mycelia of C. militaris were grown and tightened with the biochar surface. The pores generated space for the mycelia to gather nutrients in solid media and provided a carbon source for C. militaris, which was a critical nutrient for fungal growth. Thus, increasing the space area for the mycelia and carbon source affected the yields of the biomasses and cordycepin contents. Therefore, the value of adding agricultural residues is promising for the cultivation of cordycepin and higher cordycepin production. Additionally, the pyrolysis of crop wastes to biochar materials is helpful for reducing the air pollution from the dispersal of agricultural residues. These materials are plentiful, their cost is low, and the physical condition requirements are not expensive. This paper is the first to report the use of biochar for culture media for C. militaris.

5. Conclusions

In this study, the effect of media on fruiting body growth and the cordycepin production of C. militaris was investigated by mixing rice berries with different masses of corncob biochar: 0.1, 0.3, 0.5, 1, 5, and 10 g. The optimal medium contained 10 g of biochar on the rice berry in a 250 mL cylindrical glass bottle for 50 days. Using these conditions, the maximum production of cordycepin was 20.05 mg/g and 4.30 g/kg for adenosine. This method provided an effective way for enhancing cordycepin production in the solid medium, and could scale up the process for producing cordycepin contents. Moreover, this was the first time biochar was used as an additive material to improve C. militaris fruiting body and cordycepin productions, which could have a potentially positive impact on the economy and be environmentally friendly.