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The influence of environmental conditions on polysaccharide formation by Ganoderma lucidum in submerged cultures
Process Biochemist O, Vol. 33, No. 5, pp. 547-553, 1998 ~) 1998 Elsevier Science Ltd. All rights reserved Printed in Great Brilain I)032-9592/98 $19,{}{I+ 0.(1{I
ELSEVIER PII:
S0032-9592(98)00023-5
The influence of environmental conditions on polysaccharide formation by Ganoderma lucidum in submerged cultures Fan-Chiang Yang* and Chun-Bun Liau Department of Chemical Engineering, Tunghai University, Taichung, Taiwan 40704, R.().(" (Received 16 September 1997; revised version received 13 JanuaD' 1998: accepted 31 January 1998)
Abstract
The effects of environmental parameters on polysaccharide formation by Ganoderma hlcidum were investigated in submerged cultures. The optimal temperature and pH was 30-35°C and 4-4.5, respectively, in a glucose-ammonium chloride medium and polysaccharide concentration reached 1'6 mg/ml. Agitation and aeration influenced the formation and secretion of polysaccharide. The optimal rotating speed was 150 rpm in 7-day flask cultures, while the agitation speed employed in fermenter culture greatly affected the production rate and maximum concentration of polysaccharide. Although higher speeds enhanced mixing efficiency and polysaccharide release, higher shear stress had a detrimental effect on mycelial growth and polysaccharide formation. © 1998 Elsevier Science Ltd. All rights reserved K~3,words: Ganoderma lucidum, polysaccharide, submerged culture.
bility and in the degree and nature of their side-chains. Material from G. lucidum showed high activity at a dosage of 10mg/kg against Sarcoma 180 tumour in mice [4]. Activity was higher than that of other fungal fi-glucans, which possessed a high degree of branching. Because of its perceived health benefits, Lingzhi has gained wide popularity as a health food, in both Japan and China. The 1988 production of Reishi in Japan was estimated to be about 250 tons dry weight [1]. Lingzhi have normally been produced in solid cultures using substrates such as grain, sawdust or wood. Because it usually takes several months to culture the fruiting body of Lingzhi, many attempts are being made to obtain useful cellular materials or to produce effective substances from cultured mycelia [2,3]. According to the review paper of Margaritis and Pace [5], the growth and production of exopolysaccharides by microorganisms are determined by a wide range of environmental parameters, in addition to the effects of the culture medium. The objective of this work was to evaluate the effects of environmental factors on the formation of polysaccharide by G. hwidum in batch cultures [6].
Introduction
Ganoderma lucidum (Ft.) Krast (Polyporaceae) and related species are fungi used in traditional Chinese medicine. Its fruiting body is called 'Reishi' or 'Mannentake' in Japanese and 'Lingzhi' in China. In regions of China, Japan, Korea and Taiwan, Lingzhi has been a popular folk or oriental medicine used to treat various human diseases, such as hepatitis, hypertension, hypercholesterolemia and gastric cancer. Recent studies on this fungus have demonstrated many interesting biological activities, including antitumour, and anti-inflammatory effects and cytotoxicity to hepatoma cells. These studies also suggested that the carcinostatic substance in Lingzhi is a polysaccharide, 1, 3-fl-D-glucan. This polysaccharide seems to have promise as a new type of carcinostatic agent which might be useful in immmunotherapy [1-3]. A feature of a number of 1, 3-fl-D-glucans of fungal origin is their antitumour activity. The polysaccharides which demonstrate this activity are all glucans which are closely related in their structure to scleroglucan, but vary in their water solu-
*Author to whom correspondence should be addressed. 547
548
E-C. Yangand C.-B. Liau
Materials and methods
Organism Ganoderma lucidum CCRC 36123 was obtained from the Culture Collection and Research Center (CCRC), Food Industry Research and Development Institute (Hsinchu, Taiwan). Cultures were maintained on potato-agar-dextrose slopes. Slopes were inoculated and incubated at 30°C for 7 days, and stored at 4°C.
sediment three times with water, and drying to constant weight. All supernatants were collected, and then the crude polysaccharide was precipitated with the addition of 4vol of 95% ethanol. The precipitated polysaccharide was collected by centrifugation at 3000 rpm for 10 min and then dried to remove residual ethanol at 60°C. Total polysaccharide in the culture medium was determined by phenol-sulphuric acid assay according to Dubois et al. [7, 8].
Shake flask culture
Results and discussion
Shake flask cultures were carried out in 250 or 500 ml Erlenmeyer flasks containing 100 ml of medium. The media were made up of the following components (in g/l): glucose, 50; K2HPO4 0"5; KH2PO4 0"5; MgSO4 7H20 0.5; yeast extract 1; and ammonium chloride 4. Media were sterilized at 120°C for 20 min and glucose was autoclaved separately. The pH was adjusted to the desired value by addition of either 0.1 N HCI or 2.5 M NaOH. The flasks were incubated on a New Brunswick rotary shaker (Model G24) under specified conditions for 7 or 14 days.
Batch .fermentation
Jar-fermenter culture Jar-fermenter cultures were carried out in a 2-1 fermenter (Bioflo Model C32, New Brunswick Scientific) with a working volume of 1 1 to investigate the effects of aeration and agitation on the production of polysaccharide. One hundred ml of a 7 days culture was inoculated into 11 of glucose-ammonium chloride medium in a jar-fermenter, and the cells cultivated at 30°C. A six-blade impeller was used for agitation and the pH was uncontrolled in this fermenter. To study the effect of pH control, another 5-1 fermenter (CBS model CMF-5, Taipei) with a working volume of 31 was used and the pH of the broth was controlled by the automatic addition of titrants (1 M NaOH and 1 M H2804) once predetermined set point was exceeded. The fermenter was controlled at 30°C with 1 vvm aeration and agitation 100 rpm.
Analytical methods The pH was measured with a digital pH meter (Suntex, Taiwan, model 2000A). Due to the fact that mycelia and cell-bound polysaccharide could not be thoroughly separated by centrifugation, samples were first subjected to ultrasonication for 2 h in a Branson ultrosonicator (model 5210), in order to determine the concentrations of mycelium and polysaccharide. Centrifugation was then performed to remove cells and cell debris in a centrifuge (Hettich, model ERA3S/10 ml). Dry weights of total cell mass were obtained by centrifuging samples at 3000 rpm for 10 rain, washing the
Batch fermentation of G. lucidum for the production of polysaccharides was carried out in Erlenmeyer flasks and in the fermenter. The time course data of polysaccharide formation in Erlenmeyer flasks are shown in Fig. 1. The maximum concentration of the polysaccharide obtained was around 1.6 mg/ml. The difference between the levels of the maximum polysaccharide concentration obtained in shake flasks and in the fermenter was not significant (see Fig. 6). However, the production rate of polysaccharide varied widely and was greatly dependent on culture conditions.
Effect of initial pH The results from a series of experiments in Erlenmeyer flasks for 7 and 14 days are shown in Fig. 2. At very low initial pH values, such as at pH 3.0, the polysaccharide production rates were very low, reaching a concentration of 0.65 mg/ml in 14 days. At a higher initial pH value, such as at pH 4.0, the maximum polysaccharide concentrations obtained were much higher, up to 1.52 mg/ml. The highest concentration of polysaccharide was obtained at pH values of 4.0 and 4.5 in glucose-ammonium chloride medium. The concentration dropped sharply out of this range. It is useful to note that below 4.0 and above 4.5 polysaccharide concentration would decrease between 7 and 14-day culture. This suggests that the polysaccharide could be decomposed under some unfavourable conditions during culture.
Effect of temperature The result in Fig. 3 shows that the optimal temperature for polysaccharide formation by G. lucidum in glucoseammonium chloride medium was found to be 30-35°C. This range seems to be suitable for growth of mycelia and polysaccharide production [6]. The production rate of polysaccharide decreased rapidly above and below these values.
Polysaccharide .formation by G. Lucidurn in suhmerk,ed cultures
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on the formation
of polysaccharide
Effect of suface-aeration
The response of G. lucidum to intermediate levels of dissolved oxygen was investigated using shake flask cultures. Different levels of oxygen concentration and oxygen transfer were simulated by filling the same volume medium (100 ml) in 250 and 500 ml Erlenmeyer flasks (with or without baffle), which were shaken in a standard manner. It was expected that a 500 ml baffled flask would increase the surface available for oxygen transfer into the medium at the same shaker speed. The results in Table 1 show that a high oxygen transfer rate favoured polysaccharide formation and the best yield of 1.71 mg/ml was achieved when a Table I. Effects of surface aeration on the polysaccharide by G. lucidum in flask cultures Flask no.
Polysaccharide
production
cont. (mg/ml) I .08
I:;
1.22 1.62
I:;
I.71
Flask no: (I) The medium of 100 ml in a 250 ml flask without baffle; (2) The medium of 100 ml Erlenmeyer flask with baffle; (3) The medium of 500 ml Erlenmeyer flask without baffle; (4) The 100 ml in a 500 ml Erlenmeyer flask with baffle. Under the conditions of intitial pH 4, 30°C and 7 days.
of
Final pH 3.54 3.46 3.27 3.08 Erlenmeyer in a 250 ml 100 ml in a medium of 100 rpm for
by G. lucidum in 500 ml Erlenmeyer
flask at 100 rpm
baffled 500 ml Erlenmeyer flask was used in a 7 days fermentation. In all the Erlenmeyer flasks, the fermentations were subjected to oxygen limiting conditions. It should be noted that the limitation of oxygen transfer during the fermentation was also due to the increase of viscosity of liquid resulting from the accumulation of extracellular polysaccharide. Even though, the oxygen transfer coefficient was not measured during this study, this parameter should decrease significantly during the course of the fermentation. Effect of aeration rate on the formation of polysaccharide was further investigated using a jar fermenter.
Effect of aeration rate
Figure 4 illustrates the time course of polysaccharide formation at aeration rates of 1.0 and 1.5 wm. Higher aeration rate seemed to cause rapid formation of polysaccharide at earlier stage and maximum concentration was achieved in 5 days. The polysaccharide concentration at prolonged fermentation time depended strongly on the aeration rate. It was surprising to find that the results of the fermenter culture were no better than those of flask cultures. This could be attributed to the higher shear stress present in fermenter cultures, which had a negative effect on mycelia growth and polysaccharide formation.
Polysaccharide.formation by G. Lucidum in submerged cuhures Effect of rotating speed
rose to around 1'6 mg/ml in 3 days, and then, thc polysaccharide apparently gradually decomposed.
It has been observed that during the period of submerged culture of G. lucidum, the effective viscosity of the culture media increased substantially due to growth of mycelia and the accumulation of extracellular biopolymer. Efficient mixing would be vital to enhance polysaccharide synthesis and release. The influence of rotating speed on polysaccharide formation was studied on a rotary shaker in the range of 50-250 rpm in 7-day flask cultures. It has been observed that newly formed polysaccharide would stick to the mycelia pellet and this probably slowed the secretion of more polysaccharide into the media. In contrast to an optimum rotating speed of 100 rpm for mycelium growth [6], the best yield shown in Fig. 5 was achieved at a rotation speed of 150 rpm. It was believed that higher rotation speed would be favourable to the release of exopolysaccharide into the medium. However, when the rotation speed was higher than 150rpm, the yields decreased and this could be attributed to a detrimental effect of increased shear stress on the mycelium. The effect of agitation speed on polysaccharide formation in fermenter cultures is shown in Figs 6 and 7. Clearly, high agitation rate caused rapid formation of polysaccharide in the early stages. At an agitation speed of 400rpm, the polysaccharide concentration
1.60
According to the review paper of Margaritis and Pace [5], the control of pH of the broth has a great influence on the formation of microbial exopolysaccharide. Early work on xanthan fermentation demonstrated the beneficial effects of pH control on the formation of charged polysaccharide. In this test the pH was controlled at the initial value of 4 in a jar-fermenter culture. It is surprising to find that compared with the results of culture without pH control, the control had a negative effect on polysaccharide formation by G. lucidum and the level of polysaccharide decreased by 30% in a 7-day culture. Further study seems to be necessary to determine the optimal strategy for pfl control. Conclusion
Control of environmental conditions is critical in achieving higher concentration or yield of microbial products. Cell growth and production of exopolysaccharides by G. lucidum were determined over a wide range of environmental parameters. The viscosity of the broth increased significantly during the sub-
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Polysaccharide formation by G. Lucidum in .s'uhme~ed cultures
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Acknowledgements The authors wish to thank the National Science Council of R.O.C. for financial supports (NSC 85-2214-E-029-004).
References 1. Mizuno, T., Wang, G., Zhang, J., Kawagishi, H., Nishitoba, T. and Li, J., Reishi, Ganoderma lucidum and Ganoderma tsugae: Bioactive substances and medicinal effects. Food Reviews International 1995, 11(1), 151-166.
2. Sone, Y., Okuda, R., Wada, N., Kishida, E. and Misaki, A., Structure and antitumor activities of the polysaccharide isolated from fruiting body and the growing culture of mycelium of Ganoderma lucidum. Agricultural and Biological Chemisto' 1985, 49(9), 2641-2653. 3. Tscng, T. C., Shiao, M. S., Shieh, Y. S. and Had, Y. Y., Study on Ganoderma lucidum 1. Liquid culture and chemical composition of mycelium. Botanical Bulletin of Academia Sinica 1984, 25, 149-157. 4. Sutherland, I. W., Biotechnol(~o, 01' Microbial Exopolysaccharides. Cambridge University Press,New York, 1990, pp. 145-146. 5. Margaritis, A. and Pace, G. W., Microbial polysaccharides. In Comprehensive Biotechnology, Vol. 3, eds M.-Y. Murrsy, H. W. Blanch,, S. Drew. and D. I.C. Wang. Pergamon Press, Oxford, 1985, pp. 1005-1044. 6. Liau, C. B., M. Sci. thesis, Department of Chemical Engineering, Tunghai University, Taiwan, 1996. 7. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A. and Smith, F., Colorimetric method for determination of sugars and related substances. Analytical Chemistly 1956, 28(3), 350-356. 8. Pazur, J. H., Neutral polysaccharidc. In Carbohydrate Analysis - - A Practical Approach, eds M. F. Chaplin and J. F. Kennedy, Oxford IRL Press, 1987, pp. 55-142.
ELSEVIER PII:
S0032-9592(98)00023-5
The influence of environmental conditions on polysaccharide formation by Ganoderma lucidum in submerged cultures Fan-Chiang Yang* and Chun-Bun Liau Department of Chemical Engineering, Tunghai University, Taichung, Taiwan 40704, R.().(" (Received 16 September 1997; revised version received 13 JanuaD' 1998: accepted 31 January 1998)
Abstract
The effects of environmental parameters on polysaccharide formation by Ganoderma hlcidum were investigated in submerged cultures. The optimal temperature and pH was 30-35°C and 4-4.5, respectively, in a glucose-ammonium chloride medium and polysaccharide concentration reached 1'6 mg/ml. Agitation and aeration influenced the formation and secretion of polysaccharide. The optimal rotating speed was 150 rpm in 7-day flask cultures, while the agitation speed employed in fermenter culture greatly affected the production rate and maximum concentration of polysaccharide. Although higher speeds enhanced mixing efficiency and polysaccharide release, higher shear stress had a detrimental effect on mycelial growth and polysaccharide formation. © 1998 Elsevier Science Ltd. All rights reserved K~3,words: Ganoderma lucidum, polysaccharide, submerged culture.
bility and in the degree and nature of their side-chains. Material from G. lucidum showed high activity at a dosage of 10mg/kg against Sarcoma 180 tumour in mice [4]. Activity was higher than that of other fungal fi-glucans, which possessed a high degree of branching. Because of its perceived health benefits, Lingzhi has gained wide popularity as a health food, in both Japan and China. The 1988 production of Reishi in Japan was estimated to be about 250 tons dry weight [1]. Lingzhi have normally been produced in solid cultures using substrates such as grain, sawdust or wood. Because it usually takes several months to culture the fruiting body of Lingzhi, many attempts are being made to obtain useful cellular materials or to produce effective substances from cultured mycelia [2,3]. According to the review paper of Margaritis and Pace [5], the growth and production of exopolysaccharides by microorganisms are determined by a wide range of environmental parameters, in addition to the effects of the culture medium. The objective of this work was to evaluate the effects of environmental factors on the formation of polysaccharide by G. hwidum in batch cultures [6].
Introduction
Ganoderma lucidum (Ft.) Krast (Polyporaceae) and related species are fungi used in traditional Chinese medicine. Its fruiting body is called 'Reishi' or 'Mannentake' in Japanese and 'Lingzhi' in China. In regions of China, Japan, Korea and Taiwan, Lingzhi has been a popular folk or oriental medicine used to treat various human diseases, such as hepatitis, hypertension, hypercholesterolemia and gastric cancer. Recent studies on this fungus have demonstrated many interesting biological activities, including antitumour, and anti-inflammatory effects and cytotoxicity to hepatoma cells. These studies also suggested that the carcinostatic substance in Lingzhi is a polysaccharide, 1, 3-fl-D-glucan. This polysaccharide seems to have promise as a new type of carcinostatic agent which might be useful in immmunotherapy [1-3]. A feature of a number of 1, 3-fl-D-glucans of fungal origin is their antitumour activity. The polysaccharides which demonstrate this activity are all glucans which are closely related in their structure to scleroglucan, but vary in their water solu-
*Author to whom correspondence should be addressed. 547
548
E-C. Yangand C.-B. Liau
Materials and methods
Organism Ganoderma lucidum CCRC 36123 was obtained from the Culture Collection and Research Center (CCRC), Food Industry Research and Development Institute (Hsinchu, Taiwan). Cultures were maintained on potato-agar-dextrose slopes. Slopes were inoculated and incubated at 30°C for 7 days, and stored at 4°C.
sediment three times with water, and drying to constant weight. All supernatants were collected, and then the crude polysaccharide was precipitated with the addition of 4vol of 95% ethanol. The precipitated polysaccharide was collected by centrifugation at 3000 rpm for 10 min and then dried to remove residual ethanol at 60°C. Total polysaccharide in the culture medium was determined by phenol-sulphuric acid assay according to Dubois et al. [7, 8].
Shake flask culture
Results and discussion
Shake flask cultures were carried out in 250 or 500 ml Erlenmeyer flasks containing 100 ml of medium. The media were made up of the following components (in g/l): glucose, 50; K2HPO4 0"5; KH2PO4 0"5; MgSO4 7H20 0.5; yeast extract 1; and ammonium chloride 4. Media were sterilized at 120°C for 20 min and glucose was autoclaved separately. The pH was adjusted to the desired value by addition of either 0.1 N HCI or 2.5 M NaOH. The flasks were incubated on a New Brunswick rotary shaker (Model G24) under specified conditions for 7 or 14 days.
Batch .fermentation
Jar-fermenter culture Jar-fermenter cultures were carried out in a 2-1 fermenter (Bioflo Model C32, New Brunswick Scientific) with a working volume of 1 1 to investigate the effects of aeration and agitation on the production of polysaccharide. One hundred ml of a 7 days culture was inoculated into 11 of glucose-ammonium chloride medium in a jar-fermenter, and the cells cultivated at 30°C. A six-blade impeller was used for agitation and the pH was uncontrolled in this fermenter. To study the effect of pH control, another 5-1 fermenter (CBS model CMF-5, Taipei) with a working volume of 31 was used and the pH of the broth was controlled by the automatic addition of titrants (1 M NaOH and 1 M H2804) once predetermined set point was exceeded. The fermenter was controlled at 30°C with 1 vvm aeration and agitation 100 rpm.
Analytical methods The pH was measured with a digital pH meter (Suntex, Taiwan, model 2000A). Due to the fact that mycelia and cell-bound polysaccharide could not be thoroughly separated by centrifugation, samples were first subjected to ultrasonication for 2 h in a Branson ultrosonicator (model 5210), in order to determine the concentrations of mycelium and polysaccharide. Centrifugation was then performed to remove cells and cell debris in a centrifuge (Hettich, model ERA3S/10 ml). Dry weights of total cell mass were obtained by centrifuging samples at 3000 rpm for 10 rain, washing the
Batch fermentation of G. lucidum for the production of polysaccharides was carried out in Erlenmeyer flasks and in the fermenter. The time course data of polysaccharide formation in Erlenmeyer flasks are shown in Fig. 1. The maximum concentration of the polysaccharide obtained was around 1.6 mg/ml. The difference between the levels of the maximum polysaccharide concentration obtained in shake flasks and in the fermenter was not significant (see Fig. 6). However, the production rate of polysaccharide varied widely and was greatly dependent on culture conditions.
Effect of initial pH The results from a series of experiments in Erlenmeyer flasks for 7 and 14 days are shown in Fig. 2. At very low initial pH values, such as at pH 3.0, the polysaccharide production rates were very low, reaching a concentration of 0.65 mg/ml in 14 days. At a higher initial pH value, such as at pH 4.0, the maximum polysaccharide concentrations obtained were much higher, up to 1.52 mg/ml. The highest concentration of polysaccharide was obtained at pH values of 4.0 and 4.5 in glucose-ammonium chloride medium. The concentration dropped sharply out of this range. It is useful to note that below 4.0 and above 4.5 polysaccharide concentration would decrease between 7 and 14-day culture. This suggests that the polysaccharide could be decomposed under some unfavourable conditions during culture.
Effect of temperature The result in Fig. 3 shows that the optimal temperature for polysaccharide formation by G. lucidum in glucoseammonium chloride medium was found to be 30-35°C. This range seems to be suitable for growth of mycelia and polysaccharide production [6]. The production rate of polysaccharide decreased rapidly above and below these values.
Polysaccharide .formation by G. Lucidurn in suhmerk,ed cultures
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TIME (days) Fig. 1. Time course of the polysaccharidc formation by G. htcidum in 500 rnl Erlenrneyer flask at I()0 rprn, 30°( ` and initial pH 4.
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0
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Initial pH Fig. 2. Effect of initial pH on the formation of polysaccharidc by G. hu'idum in 500 ml Erlenrncyer flask at 10r) rpm and 30°( ".
E-C. Yang and C.-B. Liau
550
._.-
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Temperature (OC) Fig. 3. Effect of cultivating temperature and initial pH 4 for 7 days.
on the formation
of polysaccharide
Effect of suface-aeration
The response of G. lucidum to intermediate levels of dissolved oxygen was investigated using shake flask cultures. Different levels of oxygen concentration and oxygen transfer were simulated by filling the same volume medium (100 ml) in 250 and 500 ml Erlenmeyer flasks (with or without baffle), which were shaken in a standard manner. It was expected that a 500 ml baffled flask would increase the surface available for oxygen transfer into the medium at the same shaker speed. The results in Table 1 show that a high oxygen transfer rate favoured polysaccharide formation and the best yield of 1.71 mg/ml was achieved when a Table I. Effects of surface aeration on the polysaccharide by G. lucidum in flask cultures Flask no.
Polysaccharide
production
cont. (mg/ml) I .08
I:;
1.22 1.62
I:;
I.71
Flask no: (I) The medium of 100 ml in a 250 ml flask without baffle; (2) The medium of 100 ml Erlenmeyer flask with baffle; (3) The medium of 500 ml Erlenmeyer flask without baffle; (4) The 100 ml in a 500 ml Erlenmeyer flask with baffle. Under the conditions of intitial pH 4, 30°C and 7 days.
of
Final pH 3.54 3.46 3.27 3.08 Erlenmeyer in a 250 ml 100 ml in a medium of 100 rpm for
by G. lucidum in 500 ml Erlenmeyer
flask at 100 rpm
baffled 500 ml Erlenmeyer flask was used in a 7 days fermentation. In all the Erlenmeyer flasks, the fermentations were subjected to oxygen limiting conditions. It should be noted that the limitation of oxygen transfer during the fermentation was also due to the increase of viscosity of liquid resulting from the accumulation of extracellular polysaccharide. Even though, the oxygen transfer coefficient was not measured during this study, this parameter should decrease significantly during the course of the fermentation. Effect of aeration rate on the formation of polysaccharide was further investigated using a jar fermenter.
Effect of aeration rate
Figure 4 illustrates the time course of polysaccharide formation at aeration rates of 1.0 and 1.5 wm. Higher aeration rate seemed to cause rapid formation of polysaccharide at earlier stage and maximum concentration was achieved in 5 days. The polysaccharide concentration at prolonged fermentation time depended strongly on the aeration rate. It was surprising to find that the results of the fermenter culture were no better than those of flask cultures. This could be attributed to the higher shear stress present in fermenter cultures, which had a negative effect on mycelia growth and polysaccharide formation.
Polysaccharide.formation by G. Lucidum in submerged cuhures Effect of rotating speed
rose to around 1'6 mg/ml in 3 days, and then, thc polysaccharide apparently gradually decomposed.
It has been observed that during the period of submerged culture of G. lucidum, the effective viscosity of the culture media increased substantially due to growth of mycelia and the accumulation of extracellular biopolymer. Efficient mixing would be vital to enhance polysaccharide synthesis and release. The influence of rotating speed on polysaccharide formation was studied on a rotary shaker in the range of 50-250 rpm in 7-day flask cultures. It has been observed that newly formed polysaccharide would stick to the mycelia pellet and this probably slowed the secretion of more polysaccharide into the media. In contrast to an optimum rotating speed of 100 rpm for mycelium growth [6], the best yield shown in Fig. 5 was achieved at a rotation speed of 150 rpm. It was believed that higher rotation speed would be favourable to the release of exopolysaccharide into the medium. However, when the rotation speed was higher than 150rpm, the yields decreased and this could be attributed to a detrimental effect of increased shear stress on the mycelium. The effect of agitation speed on polysaccharide formation in fermenter cultures is shown in Figs 6 and 7. Clearly, high agitation rate caused rapid formation of polysaccharide in the early stages. At an agitation speed of 400rpm, the polysaccharide concentration
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According to the review paper of Margaritis and Pace [5], the control of pH of the broth has a great influence on the formation of microbial exopolysaccharide. Early work on xanthan fermentation demonstrated the beneficial effects of pH control on the formation of charged polysaccharide. In this test the pH was controlled at the initial value of 4 in a jar-fermenter culture. It is surprising to find that compared with the results of culture without pH control, the control had a negative effect on polysaccharide formation by G. lucidum and the level of polysaccharide decreased by 30% in a 7-day culture. Further study seems to be necessary to determine the optimal strategy for pfl control. Conclusion
Control of environmental conditions is critical in achieving higher concentration or yield of microbial products. Cell growth and production of exopolysaccharides by G. lucidum were determined over a wide range of environmental parameters. The viscosity of the broth increased significantly during the sub-
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Acknowledgements The authors wish to thank the National Science Council of R.O.C. for financial supports (NSC 85-2214-E-029-004).
References 1. Mizuno, T., Wang, G., Zhang, J., Kawagishi, H., Nishitoba, T. and Li, J., Reishi, Ganoderma lucidum and Ganoderma tsugae: Bioactive substances and medicinal effects. Food Reviews International 1995, 11(1), 151-166.
2. Sone, Y., Okuda, R., Wada, N., Kishida, E. and Misaki, A., Structure and antitumor activities of the polysaccharide isolated from fruiting body and the growing culture of mycelium of Ganoderma lucidum. Agricultural and Biological Chemisto' 1985, 49(9), 2641-2653. 3. Tscng, T. C., Shiao, M. S., Shieh, Y. S. and Had, Y. Y., Study on Ganoderma lucidum 1. Liquid culture and chemical composition of mycelium. Botanical Bulletin of Academia Sinica 1984, 25, 149-157. 4. Sutherland, I. W., Biotechnol(~o, 01' Microbial Exopolysaccharides. Cambridge University Press,New York, 1990, pp. 145-146. 5. Margaritis, A. and Pace, G. W., Microbial polysaccharides. In Comprehensive Biotechnology, Vol. 3, eds M.-Y. Murrsy, H. W. Blanch,, S. Drew. and D. I.C. Wang. Pergamon Press, Oxford, 1985, pp. 1005-1044. 6. Liau, C. B., M. Sci. thesis, Department of Chemical Engineering, Tunghai University, Taiwan, 1996. 7. Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A. and Smith, F., Colorimetric method for determination of sugars and related substances. Analytical Chemistly 1956, 28(3), 350-356. 8. Pazur, J. H., Neutral polysaccharidc. In Carbohydrate Analysis - - A Practical Approach, eds M. F. Chaplin and J. F. Kennedy, Oxford IRL Press, 1987, pp. 55-142.