Biochemical Engineering Journal Volume 10, Issue 1, February 2002, Pages 6165

Short communication

Submerged fermentation of higher fungus Ganoderma lucidum for production of valuable bioactive metabolitesganoderic acid and polysaccharide

Qing-Hua Fang, Jian-Jiang Zhong

State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China Accepted 29 August 2001. Available online 24 January 2002. http://dx.doi.org/10.1016/S1369-703X(01)00158-9, How to Cite or Link Using DOI Permissions & Reprints

Abstract
The effects of nitrogen source and initial glucose concentration were studied in submerged fermentation of higher fungus Ganoderma lucidum for simultaneous production of bioactive ganoderic acid and polysaccharide. The cells could not grow well when either yeast extract or peptone was used as the sole nitrogen source. However, a combined addition of 5 g/l of yeast extract and 5 g/l of peptone was optimal for the cell growth and metabolite production. Initial glucose concentration within 2065 g/l also greatly affected the cell growth and product biosynthesis. The extracellular polysaccharide production was remarkably improved at a high

initial glucose concentration. The highest levels of cell density (16.7 g DW/l), intracellular polysaccharide (1.19 g/l) and ganoderic acid (212.3 mg/l) were obtained at an initial glucose concentration of 50 g/l.

Keywords

Ganoderma lucidum; Ganoderic acid; Glucose and nitrogen; Higher fungi; Polysaccharide; Submerged fermentation

1. Introduction
Higher fungi are abundant sources of a wide range of useful natural products. However, compared to various researches on fermentation of conventional filamentous microorganisms (streptomycetes and fungi), a literature survey indicates that there are few investigations on the development of higher fungi bioprocesses. Ganoderma lucidum (Leyss.: Fr.) Karst, a mushroom-like fungus, is one of the most famous traditional Chinese medicinal herbs. Because of its high medicinal value, G. lucidum has received wide popularity as a health food and medicine in the Far East for a long time. Polysaccharide produced by G. lucidum is a type of carcinostatic agent, which has antitumor [1] and hypoglycemic activities [2]. The higher fungus can also produce many species of oxygenated triterpenes (especially ganoderic acid) with various biological functions such as cytotoxicity to hepatoma cells, inhibition of cholesterol synthesis and absorption, as well as stimulation of platelet aggregation [3]. Because it usually takes several months to cultivate the fruiting body of the fungus and it is also difficult to control the product quality during its cultivation, there is a great need to supply the market with high-quality G. lucidum products. In contrast, submerged fermentation of G. lucidum is viewed as a promising alternative for efficient production of its valuable metabolites polysaccharide [1], [4] and [5] and ganoderic acid [6]. Until now, there is lack of reports on the simultaneous production of polysaccharide and ganoderic acid by fermentation technology. Both nitrogen and carbohydrates are major nutrient sources in a culture process. In fermentation of G. lucidum, there is no information available regarding the influences of nitrogen source and initial glucose concentration on the accumulation of ganoderic acid and polysaccharide. In this article, we report the production of these two valuable metabolites by submerged fermentation of G. lucidum in shake flasks. The effects of nitrogen source and initial glucose concentration were investigated in order to improve the cell growth and the production of polysaccharide and ganoderic acid. The relationship between the pellet morphology and production of polysaccharide and ganoderic acid

was also studied. The information obtained in this work is considered fundamental and useful to the further development of the higher fungus fermentation process on a bioreactor scale.

2. Materials and methods

2.1. Maintenance and preculture of G. lucidum
The strain of G. lucidum CCGMC 5.616 was purchased from China General Microbiological Fermentation Collection Center (Beijing, China). It was maintained on potato-agar-dextrose slants. The slant was inoculated and incubated at 28 C for 7 days, then stored at 4 C for about 2 weeks. Preculture medium consisted of the following components: sucrose 35 g/l, peptone 5 g/l, yeast extract 2.5 g/l, KH2PO4H2O 1 g/l, MgSO47H2O 0.5 g/l, and Vitamin B1 0.05 g/l. For the first preculture, 40 ml medium with initial pH of 5.5 was prepared in a 250 ml flask, and then 10 ml mycelium suspension from a slant culture was inoculated, and followed by 7 days incubation at 30 C on a rotary shaker (120 rpm). For the second preculture, 45 ml medium was prepared in a 250 ml flask, and inoculated with 5 ml first preculture broth (with ca. 100 mg DW of cells/l), then followed by 4 days incubation at 30 C on a rotary shaker (120 rpm).

2.2. Nitrogen source and initial glucose concentration

The effect of nitrogen source on the fungus culture was studied by using various organic and inorganic nitrogen sources, i.e. 1.25 g/l of yeast and 1.25 g/l of peptone, 2.5 g/l of yeast extract, 2.5 g/l of peptone, 2.5 g/l of casein, 2.5 g/l of NH4NO3, or 2.5 g/l of NH4Cl. The different concentrations of yeast extract and peptone were further tested to obtain an optimal cell growth and product accumulation. The fermentation medium (at an initial pH of 5.5) was composed of 35 g/l of glucose, 1 g/l of KH2PO4H2O, 0.5 g/l of MgSO47H2O, 0.05 g/l of vitamin B1, and different levels of nitrogen sources as investigated. The initial glucose concentrations tested were 20, 35, 50 and 65 g/l. The fermentation medium (at an initial pH of 5.5) was composed of 5 g/l of peptone, 5 g/l of yeast extract, 1 g/l of KH2PO4H2O, 0.5 g/l of MgSO47H2O, 0.05 g/l of vitamin B1, and a different level of glucose as investigated. The medium was inoculated by transferring 5 ml second-stage preculture broth (with ca. 300 350 mg DW of cells/l) to 45 ml medium in a 250 ml flask. The fermentation was conducted in the dark at 30 C on a rotary shaker at 120 rpm. Multiple flasks were run at the same time, and three flasks were sacrificed at each sampling point.

2.3. Determination of dry weight, pellet size, medium sugar, and oxygen uptake rate
For measurement of cell dry weight, the cells from a sample were filtered through a mesh with 30 m pore size and washed with a large amount of distilled water, then collected by centrifugation at 16,000 rpm. The fresh cells were dried at 50 C for sufficient time until a constant dry weight was obtained. After sampling, the fermentation supernatants were stored at 20 C, and then

thawed for analyses of residual glucose by glucose oxidase kit. By using a series of testing sieves, pellets from a sample were separated into different sizes and their size distribution frequency was based on the ratio of fresh cell weights. The determination of oxygen uptake rate (OUR) was conducted by conventional dynamic method (at 30 C). That is, the cells were added into a 300 ml chamber full of air-saturated water. Then, the chamber (with a DO probe) was enclosed, while the cells were kept in suspension by stirring with a magnetic bar. The decrease of DO level was recorded and OUR was estimated from the DO slope against time. Specific OUR (SOUR) was calculated from the OUR and cell dry weight.

2.4. Measurements of extracellular and intracellular polysaccharide

For the determination of extracellular polysaccharide (EPS), after removal of mycelia by filtration, the fermentation filtrate was dialyzed, and the crude extracellular polysaccharide was precipitated with addition of 95% (v/v) ethanol by four times of volume, then separated by centrifugation at 13,000 rpm. The insoluble components were suspended in 1 M NaOH at 60 C for 1 h, and the supernatant was measured by phenolsulfuric acid method [7]. For the analysis of intracellular polysaccharide (IPS), the dried mycelia (100 mg) were extracted by 1 M NaOH at 60 C (1 h), then the supernatant was assayed by phenol-sulfuric acid method [7].

2.5. Assay of ganoderic acid

The determination of ganoderic acid (GA) content was similar to that as described by Tsujikura et al. [6]. The dried mycelia (100 mg) were extracted by 50% (v/v) ethanol (3 ml) for one week (twice). After removal of mycelia by centrifugation, the supernatants were dried at 50 C under vacuum. The residues were suspended by water, and later extracted with chloroform. The ganoderic acid in chloroform was extracted by 5% (w/v) NaHCO3. After adding 2 M HCl to adjust the pH of the NaHCO3 layer to be lower than 3, the ganoderic acid in the NaHCO3 layer was extracted with chloroform. After removal of chloroform by evaporation at 40 C, ganoderic acid was dissolved in absolute ethanol, and its absorbency was detected at 245 nm.

3. Results and discussion

3.1. Effect of nitrogen sources
Compared with inorganic nitrogen sources (NH4NO3 and NH4Cl), when organic ones (yeast extract, peptone, casein) were used, the cells grew more quickly and reached a higher final cell density by DW (Fang, Q.H., MS thesis, ECUST, 2000). The highest cell concentration of 4.7 g DW/l and an average growth rate of 2.44 per day were obtained, when a combination of 1.25 g/l of yeast extract and 1.25 g/l of peptone was used. The results suggest that certain essential amino acid(s) could not be synthesized from inorganic nitrogen sources by the cultured cells of the higher fungus. As reported, organic nitrogen source was used in almost all the cases of G. lucidum fermentations [1], [4] and [5].

In order to obtain a higher dry cell weight, we attempted to optimize the initial concentrations of peptone and yeast extract by altering their addition levels (Table 1). Under various initial levels of peptone at 2.5 g/l of yeast extract, the maximum cell density at a peptone concentration of 0, 2.5, 5, and 10 g/l was 4.2, 10.8, 12.7 and 8.2 g DW/l, respectively. Under various initial levels of yeast extract at 5 g/l of peptone, the maximum cell density at a yeast extract concentration of 0, 2.5, 5 and 10 g/l was 4.1, 10.7, 15.4 and 15.8 g DW/l, respectively. It was found that the yield of cells upon protein was low and medium glucose was not fully consumed (data not shown), when either peptone or yeast extract was used as the sole nitrogen source. But, when both of them were used, glucose was fully utilized and the growth yield value was high (data not shown). These results suggest that yeast extract and peptone had complementary effects in promoting the cell growth. However, when a relatively high level of peptone (10 g/l) was added, the cell growth was inhibited and the growth yield value was also decreased. The reason was supposed to be that there may exist certain growth inhibitor(s) in peptone and the inhibitory effect surpassed the stimulating effect of peptone at a higher concentration. In addition, the highest cell concentration of 15.8 g DW/l obtained under 5 g/l of peptone in combination with 10 g/l of yeast extract was higher than that in previous reports [1] and [4]. Table 1. Effects of initial concentrations of peptone and yeast extract in medium on the growth and metabolite production by G. lucidum cellsa Cell dry Average growth weight (g/l) rate (per day) Peptone (g/l)b 4.20 0.1 0 1.33 (day 8)d 10.8 0.1 2.5 3.64 (day 8) 12.7 0.6 5 4.28 (day 8) 8.2 0.4 10 2.72 (day 8) Yeast extract (g/l)c 4.1 0.4 0 1.55 (day 8) 10.7 0.8 2.5 4.32 (day 10) 15.4 0.1 5 6.24 (day 8) 15.8 0.25 10 6.41 (day 8) a Maximum IPS production (g/l) 0.25 0.02 0.63 0.01 0.78 0.06 0.51 0.03 Maximum EPS production (g/l) 0.25 0.03 0.32 0.04 0.48 0.02 0.36 0.03 Maximum GA production (g/l) 33.3 3.5 110.5 10.2 101.6 4.4 84.1 6.9

0.23 0.03 0.51 0.06 0.81 0.03 0.86 0.01

0.45 0.03 0.50 0.05 0.81 0.06 0.62 0.02

42.4 1.7 112.4 19.8 170.9 17.2 150.0 14.8

Standard deviation was calculated from three independent samples. b

At 2.5g/l of yeast extract. c At 5g/l of peptone. d Culture time (days). For the production of intracellular polysaccharides (IPS) and EPS at different concentrations of yeast extract and peptone, the highest accumulation of IPS (0.86 g/l) was obtained when 10 g/l of yeast extract and 5 g/l of peptone were used (Table 1). For the EPS production, it reached 0.81 g/l when 5 g/l of yeast extract and 5 g/l of peptone were used in combination. Lee et al. [4] reported the production of extracellular polysaccharide as high as 20.04 g/l in fermentation of G. lucidum. But, as we confirmed, their measurement by gravimetric method was not accurate due to the impurity residues in the samples, and it could overestimate the polysaccharide amount by almost nine-fold of that by using a conventional phenol-sulfuric acid method as we used here. The highest accumulation of GA, i.e. 170.9 mg/l, was obtained at 5 g/l of yeast extract and 5 g/l of peptone (Table 1). The GA content reached 1.40 mg/100 mg DW, which was much higher than a previous work [6].

3.2. Effect of initial glucose concentration

As shown in Table 2, the maximum dry cell mass at an initial glucose level of 20, 35, 50 and 65 g/l was 10.8, 14.1, 16.7 and 12.6 g DW/l as obtained at days 6, 8, 10 and 10, respectively. However, the average growth rate decreased with an increase of initial glucose concentration, and it was 5.82, 5.73, 5.45 and 4.11 d1 at an initial glucose concentration of 20, 35, 50 and 65 g/l, respectively. Such a phenomenon was also claimed in Aspergillus niger cultures [8]. Patel and Agnew [9] also suggested that in the presence of higher concentration of soluble substrates (like sugar), the growth of many microorganisms was inhibited due to unfavorable osmotic pressures. In our work the osmotic pressure at 20, 35, 50 and 65 g/l of initial glucose was estimated to be 2.76, 4.83, 6.90 and 8.97 atm, respectively, according to the literature [10]. The results suggested that the cell growth was inhibited by a relatively higher initial sugar concentration due to high osmotic pressure. Table 2. Effects of initial glucose level on the cell growth, yield, and production of IPS, EPS and GAa Initial glucose concentration (g/l) 20 35 50 Cell dry weight (g/l)

65

10.8 0.35 (d 14.1 0.13 (d 16.7 0.09 (d 12.6 0.12 (d 6)b 8) 10) 10)

Yieldx/s (g DW per g of glucose) 0.577 Average growth rate (per day) 5.82

0.498 5.73

0.419 5.45

0.347 4.11 0.95 0.029 1.08 0.02 1.25 0.05 158.8 7.8

Maximum IPS production (g/l) 0.55 0.028 0.87 0.064 1.19 0.051 Maximum EPS production (g/l) 0.43 0.05 0.66 0.02 0.85 0.029 Maximum GA content 1.56 0.04 1.29 0.11 1.27 0.12 mg/100 mg DW Maximum GA production (mg/l) a 168.4 4.5 179.7 17.3 212.3 20.8

Standard deviation was calculated from three independent samples. b Culture time (days). In all the cases, the SOUR was stable around 0.62 mmol O2/(g DW h) in the first 4 days submerged culture. After that, at an initial glucose concentration of 35, 50 and 65 g/l, the SOUR increased greatly and reached the peak on days 6, 10 and 10, respectively. At an initial glucose level of 20 g/l, the SOUR increased only a little and reached the peak on day 6. The highest SOUR at an initial glucose concentration of 20, 35, 50 and 65 g/l was 0.680, 0.874, 0.893 and 0.971 mmol O2/ (g DW h), respectively. In all the cases, the SOUR showed a sharp decrease after the peak, and the pellet size also became smaller. These phenomena suggest that the cells physiology changed drastically during the later stage of fermentation. The low respiratory activity of cells at a low initial glucose level might be related with the large pellet size in the case (Fig. 1). As indicated in Table 3, a large pellet size had low SOUR, which may be due to the oxygen limitation in the pellet center. The cell yield against glucose sharply decreased with an increase of initial glucose level (Table 2), respectively. The reason was considered as the high respiration activity and more production of CO2 at a higher initial glucose concentration.

Fig. 1. Effect of initial glucose concentration on the pellet size of G. lucidum (on day 8). Symbols for pellet sizes: diameter larger than 1.6 mm (dark bar), diameter between 1.2

1.6 mm (blank bar), and diameter smaller than 1.2 mm (hatched bar). The error bars in the figure indicate the standard deviations from three independent samples. Table 3. Effect of pellet size on SOUR and content of polysaccharide and ganoderic acida Diameter of pellets Larger than Between 1.2 1.6 mm 1.6 mm SOUR (mmol O2/(g DW h) 0.65 0.02 0.86 0.03 GA content (mg/100 mg DW) 1.62 0.21 1.27 0.14 IPS content (mg/100 mg DW) 5.48 0.31 6.92 0.25 a Standard deviation was calculated from three independent samples. The pellet size was very different at a different initial glucose concentration (Fig. 1). It was found that a higher initial glucose concentration resulted in a relatively smaller pellet size. The result suggests that the pellet size might be affected by different osmotic pressure, which was caused by different initial sugar levels [10]. When the initial glucose level increased from 20 to 65 g/l, the initial osmotic pressure increased 6.21 atm. As reported in Panax notoginseng cell cultures [11], the cells were also shrunk due to high medium osmotic pressure. For the IPS content, at the end of culture (day 14), its maximum was 7.15, 7.29, 7.43 and 7.96 mg/100 mg DW at an initial glucose concentration of 20, 35, 50 and 65 g/l, respectively. The different IPS content in this experiment may be related to the different pellet size at different initial glucose concentration. At high glucose concentration, because of the high osmotic pressure, the pellet size was smaller (Fig. 1), and the small pellet was found favorable for the IPS biosynthesis (Table 3). The total accumulation of IPS reached 0.55, 0.87, 1.19 and 0.95 g/l on days 6, 8, 10 and 10 at an initial glucose level of 20, 35, 50 and 65 g/l (Table 2); and the corresponding IPS productivity was 0.090, 0.107, 0.118 and 0.093 g/l per day, respectively. These results show that initial glucose concentration of 50 g/l was favorable to both the production and productivity of IPS due to a relatively high cell density obtained under the condition. The highest EPS production at initial glucose concentration of 20, 35, 50 and 65 g/l was 0.43, 0.66, 0.85 and 1.08 g/l (Table 2) as obtained on days 8, 10, 12 and 12, respectively, and the corresponding EPS productivity was 0.050, 0.063, 0.069 and 0.087 g/l per day. The results suggest that high initial glucose concentration led to both high production and productivity of EPS. Although a low initial glucose concentration resulted in high GA content, the maximum GA production was obtained at an initial glucose concentration of 50 g/l because of the high maximum biomass obtained in this case (Table 2). The highest production (and productivity) of GA was 168.4 (28.1), 179.7 (22.5), 212.3 (21.2), and 158.8 mg/l (15.9 mg/l per day) at an initial glucose level of 20, 35, 50 and 65 g/l, respectively. The results indicated that a high cell density was desirable especially for intracellular products in order to increase the metabolite production, and the relatively high productivity of GA at a low initial glucose concentration was mainly due to the high cell growth rate in the same condition.

Smaller than 1.2 mm 1.22 0.02 0.98 0.12 8.49 0.28

Acknowledgements
The financial support from National Natural Science Foundation of China (Project No. 20076011) and Shanghai Science & Technology Commission is gratefully acknowledged. J.J.Z. also thanks the Cheung Kong Scholars Program, the Ministry of Education of China.

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