Effects of cultivating conditions on the mycelial growth of Ganoderma lucidum in submerged flask cultures F C. Yang, C B. Liau Abstract In this paper the effects of environmental con- ditions on the mycelial growth of Ganoderma lucidum were investigated in shake ¯ask cultures. The optimal temperature and pH were found to be around 30±35 °C and 4, respectively, in a glucose-ammonium chloride me- dium. The maximum mycelial concentration reached to around 350 mg/100 ml. The formation of mycelial pellets and their ultra structure was demonstrated to be greatly affected by cultivating conditions. Increasing surface ae- ration would be bene®cial for mycelial growth. However, too high rotating speed in shake cultures had a detrimental effect on the formation of mycelial pellets and the opti- mum was found to be 100 rpm. 1 Introduction Ganoderma lucidum (Fr.) Karst (Polyporaceae) is a species of basidiomycetes which belongs to polyporacceae (or Ganodermaceae) of Aphyllophorales. Its fruiting body is called ``Reishi'' in Japanese and ``Lingzhi'' in China. In the regions of China, Japan and Korea, Lingzhi has been a popular folk or oriental medicine to cure various human diseases, such as hepatitis, hypertension, hype- rcholesterlemia and gastric cancer. Recent studies on this fungus have demonstrated many interesting biological activities, including antitumour, and anti-in¯ammatory effects and cytotoxicity to hepatoma cells. These studies also suggested that the carcinostatic substance in Lingzhi is a polysaccharide, b-(1- > 3) -D-glucan [1, 2]. This polysaccharide seems to show promise as a new type of carcinostatic agent which might be useful in immuno- therapy. Lingzhi, because of its perceived health bene®ts, has gained wide popularity as a health food, in both Japan and China. Reishi cultivation has prospered in Japan, China, Taiwan, and Korea. In addition, attempts are being made to obtain useful cellular materials or to produce effective substances from cultured mycelia [1±3]. Mushroom mycelia or spawns have normally been produced in solid cultures using substrates such as grain, sawdust or wood. Propagation of edible mushrooms in submerged culture was initially developed during the 1950s based on the success of growing lower fungi in fermenters for economical production of various natural products. Since then numerous attempts have been made by researchers to cultivate mushroom mycelium com- mercially in submerged culture[4]. Submerged culture has the potential advantage in that it can be dispersed within the substrate more uniformly than solid spawn and the time taken to produce the ®rst crop of sporophores may be shortened. Further, the liquid nature of such spawn en- ables inoculation to be carried out under relatively more stringent aseptic conditions which is important when using non-selective substrates[4±6]. Yields and productivity of mushroom mycelium vary widely, depending on the mushroom, substrate, and con- ditions. Although many workers have attempted to obtain mycelium of Ganoderma lucidum using submerged cul- ture, very little information is available regarding the environmental factors affecting mycelial growth of G. lucidum in submerged culture[2, 3]. The study reported here was carried out to determine the physical conditions required for the mycelial growth of G. lucidum in sub- merged shake cultures. In this paper, we also report factors affecting the formation of mycelial pellets and their ultra structure[7]. 2 Materials and methods 2.1 Microorganism and media The culture used was Ganoderma lucidum CCRC 36123 obtained from the Culture Collection and Research Centre (CCRC), Food Industry Research and Development Insti- tute (Hsinchu, Taiwan). Culture was maintained on potato- dextrose-agear slope. Slopes were inoculated and incubated at 30 °C for 7 days, and stored at 4 °C. The media were made up of the following components (in gram per liter): glucose 50; K 2 HPO 4 0.5, KH 2 PO 4 0.5, MgSO 4 á 7H 2 O 0.5 yeast extract 1 and ammonium chloride 4. 2.2 Cultivation of microorganism The shake-¯ask experiments were performed in 500-ml Erlenmeyer ¯asks containing 100 ml of the media. Media were sterilized at 120 °C for 20 min and glucose was au- toclaved separately. The pH was measured and adjusted to Bioprocess Engineering 19 (1998) 233±236 Ó Springer-Verlag 1998 233 Received: 21 October 1997 F C. Yang, C B. Liau Department of Chemical Engineering, Tunghai University, Taichung, Taiwan 40704, R.O.C Correspondence to: F C. Yang The authors wish to thank the National Science Council of R.O.C. for ®nancial supports (NSC 85-2214-E-029-004). the desired value by addition of either 4M-HCl or 2.5M NaOH. Actively growing mycelia (4 pieces, each 5mm´ 5 mm) from a newly prepared slant culture (about 7 days incubation at 30 °C) were inoculated into the ¯ask. The ¯asks were shaken on a New Brunswick rotary shaker (Model G24) at 100 rpm and 30 °C. At the end of inoculation period mycelium consisting of individual pellets was harvested by centrifugation and wash for the analysis. The yield was expressed as mg/100 ml dry weight. The range in size of individual mycelium pellet was determined by measuring the average diameter of pellets per culture. 2.3 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 sepa- rated by centrifugation, in order to determine the con- centrations of mycelium and polysaccharide, samples were ®rst subjected to ultrasonication for 2 hrs in a Branson ultrosonicator (model 5210). 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 min, washing the sediment three times with water, and drying to constant weight. 3 Results and discussion 3.1 Effect of initial pH The mycelia of various species of mushrooms will grow over a wide range of pH values. However, for most or- ganisms, the most favorable pH range is from 5 to 7. The optimal initial pH for growth was determined for the se- lected strain in glucose-ammonium chloride medium or glucose-malt extract medium over a pH range of 3.0 to 6.0, incubating for 7 days or 14 days. The optimum pH for the highest yield of G. lucidum in a glucose-ammonium chloride medium was 4.0 as shown in Fig. 1. However, it is interesting to note that the optimal pH for G. lucidum growing in a glucose-malt extract medium was found to be around 5.0. It demonstrated that optimal initial pH for mycelial growth would depend on the culture medium. Lower values of initial pH would be bene®cial to inhibit the growth of bacterial contaminants. In general, when ammonium salts were used as the nitrogen source, the pH decreased during the mycelium growth as the result of assimilation of the ammonium ion and the attendant effects of acids anions in the medium such as chlorides, sulfate, or phosphate. The lower limit of this pH decrease depends upon the buffering action of the constituents of the medium and the mycelium[4]. 3.2 Effect of cultivating temperature A narrow temperature range for submerged culture of G. lucidum mycelia has been reported. The results in Figs. 2 and 3 show that the optimal growth temperature of Fig. 1. Effect of initial pH on the growth of mycelium of G. lucidum in the glucose-NH 4 Cl medium on a rotary incubator at 100 rpm and 30 °C Fig. 2. Effect of cultivating temperature on the growth of myce- lium of G. lucidum in the glucose-NH 4 Cl medium on a rotary incubator at 100 rpm and initial pH 4.0 Fig. 3. Effect of cultivating temperature on the growth of myce- lium of G. lucidum in the glucose-malt extract medium on a rotary incubator at 100 rpm and initial pH 5.0 234 Bioprocess Engineering 19 (1998) G. lucidum in glucose-malt extract medium or glucose- ammonium chloride medium was found to be 30±35 °C. The growth rate of this organism decreases rapidly above and below these values. The lower values of ®nal pH seem to indicate better growth of G. lucidum mycelia. 3.3 Effect of surface aeration The effects of the surface aeration were investigated by varying the volume of ¯ask so that the ratio of the surface area of the liquid exposed to air to the liquid volume was varied, affecting the aeration during the shaking process. Different volumes of Erlenmeyer ¯asks (250 ml and 500 ml) with or without baf¯e were ®lled with the medium of 100 ml and shaken in a standard manner. Table 1 shows that increased surface aeration enhanced the ®nal dry mycelium concentration obtained in the fermentations and the best yield could be achieved when a baf¯ed 500 ml Erlenmeyer ¯ask was used in a 7 days fermentation. However, the mycelium form varied. Larger pellets were produced at lower surface aeration rates, corresponding to a lower dry mycelium concentration. Smaller pellets were obtained when surface aeration rates increased, corre- sponding to a higher dry mycelium concentration. The formation of pellets is largely determined by the extent of agitation and aeration. The effect of aeration on the growth of mushroom mycelium, speci®cally in the development of a pelleted mycelium, is complex and, in some cases, contradictory. In general pellet formation is favoured by low agitation and aeration rates. The oxygen mass transfer in the fermentation suspension is enhanced when pellets are formed because of the lower resistance produced by lowering the viscosity of the medium, as compared with ®lamentous growth in the same type of medium. However, the oxygen supply to the interior of the pellets decreases because of the condensation of mycelium characteristics of the pellet structure and as a function of the pellet diameter. Diffusion of oxygen from the medium into large pellets is assumed to be the limiting factor for growth of mushroom mycelium. According to the paper of Litch®eld in 1967[4], aeration rates in the range used by other common aerobic fer- mentations are usually detrimental to mushroom myceli- um growth. In some cases of cultivating mushrooms in submerged culture, it was observed by several investiga- tors that increasing aeration rate resulted in ®lamentous growth, and reduced yield. The effect of high aeration rate on the growth of mycelium of G. lucidum should be studied further by using the fermenter. 3.4 Effect of shaking frequency The in¯uence of rotating speed on mycelium growth was studied in the range of 50±250 rpm while all other conditions were kept constant. As to the results shown in Fig. 4, the maximum concentration of mycelium was ob- served at the shaking frequency of 100 rpm. There was an increase in the yield of mycelium when the shaking fre- quency was increased from 50 to 100 rpm. It is supposed that a higher rpm implies a better oxygen transfer in the fermenting medium. However, the fact that the biomass yields were lower above 100 rpm could be attributed to a detrimental effect of increased shear stress on the myce- lium. The sizes of the pellets formed and their distributions were mainly affected by the rotating speed. As to the re- sults shown in Fig. 5, the size of mycelial pellets decreased with increase in shaking frequency. At low rotating speed, larger mycelial pellets were formed and the pellets formed were very incompact in nature and were loosely arranged. However, at higher speed, due to the excessive shear force, the pellets formed were extremely tiny in size. This may Table 1. Effects of surface aeration on the growth and ultra structure of mycelium in ¯ask cultures of G. lucidum Flask No. Mycelium conc. (mg/100 ml) Final pH Pellet number Pellet diameter (mm) (1) 144 5.05 21 10 (2) 232 4.64 9 14 (3) 479 3.97 100 3 (4) 555 3.88 150 2 1. Flask No. (1). The medium of 100 ml in 250 ml-Erlenmeyer ¯asks without baf¯e (2). The medium of 100 ml in 250 ml-Erlenmeyer ¯asks with baf¯e (3). The medium of 100 ml in 500 ml-Erlenmeyer ¯asks without baf¯e (4). The medium of 100 ml in 500 ml-Erlenmeyer ¯asks with baf¯e 2. under the conditions of initial pH = 5.6, 30 °C and 100 rpm for 7 days 3. malt extract 4 %, yeast extract 0.1 %, K 2 HPO 4 0.05 %, KH 2 PO 4 0.05 %, MgSO 4 á 7H 2 O 0.05 % Fig. 4. Effect of rotating speed on the growth of mycelial of G. lucidum in the glucose-NH 4 Cl medium on a rotary incubator at 100 rpm and 30 °C 235 indicate that G. lucidum is an obligate aerobe and the optimum rotating speed is around 100 rpm. 3.5 Effect of size of inoculum In order to enhance cell density and also to develop a method for large scale cultures, various sizes and types of inoculum were tested. Inoculum was prepared by submerged culture using a shake ¯ask for 7 days. After agitation in a blender for 5 seconds, the mycelia were inoculated into the ¯asks with 4 levels of inoculum (1, 4, 8, 12 ml per 100 ml broth). After 7-day cultures at 30 °C and 100 rpm, the ®nal mycelia concentrations were 235, 351, 462, and 579 mg per 100 ml, respectively. In contrast to inoculation from a slant, when submerged culture was used for inoculum, the mycelial pellets became smaller and more uniform in size. The results showed that an increase in inoculum concentration increased the yield of myceli- um and the number of pellet but decreased the size of mycelial pellets. However, when too many pellets were present in the broth, some tiny particles could stick to- gether during the culture and caused the increase of pellet size. 4 Conclusions As described above, yields and productivity of mushroom mycelium vary widely, depending on the mushroom, substrate, and conditions. Effects of some environmental factors on the growth of mycelia of G. lucidum were investigated in shake ¯ask culture in this report. The formation of mycelial pellets and their ultra structure was also demonstrated to be greatly affected by cultivating conditions. However, in order to meet the requirement of large scale production, further study about the effect of agitation and aeration on the growth of mycelia of G. lucidum in fermenter cultures would be necessary. References 1. 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Biotechnology and Food Ingredients, pp. 31±64, New York: Van Nostrand Reinhold 1991 6. Song, C.H.; Cho, K.Y.: A synthetic medium for the production of submerged cultures of Lentinus edodes. Mycologia. 79 (6) (1987) 866±876 7. Liau, C.B.: M. Sci. thesis, Department of Chemical Engineering, Tunghai University, Taiwan, 1996 Fig. 5. Effect of rotating speed on the ultrastructure of mycelial pellet of G. lucidum in the glucose-NH 4 Cl medium on a rotary incubator at 100 rpm and 30 °C 236 Bioprocess Engineering 19 (1998) . Effects of cultivating conditions on the mycelial growth of Ganoderma lucidum in submerged flask cultures F C. Yang, C B. Liau Abstract In this paper the effects of environmental con- ditions. when ammonium salts were used as the nitrogen source, the pH decreased during the mycelium growth as the result of assimilation of the ammonium ion and the attendant effects of acids anions in the. corre- sponding to a higher dry mycelium concentration. The formation of pellets is largely determined by the extent of agitation and aeration. The effect of aeration on the growth of mushroom