Journal of Culture Collections National Bank for Industrial Microorganisms and Cell Cultures ISSN: 1310-8360 Vol. 6, Num. 1, 2009, pp. 97-105

Journal of Culture Collections, Vol. 6, No. 1, 2009, pp. 97-105

Comparative study of environmental and nutritional factors on the mycelial growth of edible mushrooms

Ahmed Imtiaj, Chandana Jayasinghe, Geon Woo Lee and Tae-Soo Lee^*

Department of Biology, University of Incheon, Incheon 402-749, Korea
*Corresponding author, e-mail: tslee@incheon.ac.kr

Code Number: cc09012

Summary

Mushrooms are usually fleshy fungi belonging to either basidiomycota or ascomycota.The optimal culture conditions for the mycelial growth of 371 strains be-longing to 9 species of edible mushrooms were investigated. The temperaturesuitablefor the mycelial growth was found to be 25 ºC and optimal range of temperature was 20~30 ºC. Mushrooms have a broad pH range (5~9) for their mycelial growth but mostly favorable pH was between pH 6 and 7. Hamada, Lilly, PDA and YM were the most appropriate nutrient media whereas Czapek Dox, Hoppkins and Hennerberg were the most unsuitable for radial mycelial growth of mushrooms. Among 10 different carbon sources, dextrin, fructose and sucrose were the best while lactose and galactose were the most unfavorable ones. In carbon sources, mycelial density was found to vary from compact to somewhat compact. The most suitable nitrogen sources for mycelial growth were glycine, ammonium acetate and calcium nitrate whereas the most unsuitable were histidine and ammonium phosphate. In case of glycine, mycelial density was found to vary from nearly compact to compact but in the rest of nitrogen sources the mycelial density was rather thin.

Key words: culture conditions, edible mushroom,media, mycelial growth, nutrition

Introduction

Mushrooms are fleshy fungi belonging to either basidiomycota or ascomycota. Some members of mushrooms are important and delicious items of our food menu having both nutritive and medicinal values [2, 6]. This item is appreciated, not only for texture and flavor, but also for their chemical and nutritional characteristics [14]. On a dry weight basis, they are considered to be good sources of digestible proteins (10–40 %), carbohydrates (3–21 %) and dietary fiber (3–35 %). Mushrooms contain all the essential amino acids [5]. This group of fungi is an excellent source of vitamin B₁ (thiamine), B₂ (riboflavin), B₃ (nicotinic acid), C (ascorbic acid) and biotin. Edible mushrooms in cooked or other processed forms are nutritionally sound and good dietary components suitable for vegetarians, diabetic and heart patients [4]. Mushrooms have also been reported as therapeutic foods, useful in preventing diseases such as hypertension, hypercholesterolemia and cancer [3]. Some recently isolated and identified compounds, originating from mushrooms, show other significant medical properties, such as immunomodulatory, liver protecttive, antifibrotic, antiinflammatory, antidiabetic, antiviral and antimicrobial activities [10, 17, 25, 26]. The world production of different cultivated mushrooms is about 1.3 billion pounds [19] such as Agaricus bisporus 1956, Lentinula edodes 1564, Volvariella volvacea 181, Pleurotus spp. 876, Auricullaria spp. 485 and Flammulina velutipes 285 tons in 1997 [16]The production was increased for A. bisporus16.1, L. edodes 25.5, Volvariella sp.16.3, Pleurotusspp.537.9, Auricullaria spp. 336.1 and F. velutipes with 43.0 % in 1990, compared to 1986 [18]. It is generally recognized that growing mycelia in a defined medium is a rapid and alternative method to obtain fungal biomass for further use [27]. It is also well known that the mycelial growth of mushrooms is enhanced by different environmental and nutritional factors. Because of that, our study has been conducted on the mycelial growth of 371 strains belonging to 9 species of edible mushrooms. The different environmental (pH, temperature) and nutritional factors (media, carbon and nitrogen sources) were used to assess the optimal culture conditions for the mycelia of mushrooms.

Collection of strains. The cultures of 371 strains belonging to 9 species of edible mushrooms such as A. bisporus, Agrocybe cylindracea, F. velutipes, Hypsizygus marmoreus, L. ådodes, Lentinus lepideus, Pholiota adiposa, Pleurotus eryngii and Pleurotusostreatus were obtained from Culture Collection of Wild Mushroom (CCWM) species, University of Incheon, Korea. All strains obtained from CCWM were recultured on potato dextrose agar (PDA) medium and incubated for 10 days at 25 ºC for further study(Table 1). Three replications were performed for each strain in different experiments.

Table 1. List of strains used in this experiment.

*Number of studied strains per species.
Three replications were used for each strain in every experiment.

Effect of temperature. To screen the suitable temperature for the mycelial growth the 9 species of edible mushrooms were incubated for 10 days at 5 different temperatures. A 5 mm diameter agar plug removed from 10 days old cultures grown on PDA was placed in the centre of each plate filled with PDA. The medium was adjusted to pH 6 and incubated for 10 days at 15 ºC, 20 ºC, 25 ºC, 30 ºC and 35 ºC, separately. Radial growth of mycelia on each Petri dish was measured at 3 directions such as A to B, C to D and E to F (Figure 1). Average value of mycelial growth of each Petri dish was calculated out of those 3 measurements. Similarly, mycelial growth was measured of remaining 2 Petri dishes (since 3 replications were used for each strain). The following formula was used to calculate the mycelial growth of mushrooms.

Average mycelial growth on:

1^st Petri dish (AB₁+CD₁+EF₁) /3 = R₁
2^nd Petri dish (AB₂+CD₂+EF₂) /3 = R₂
3^rd Petri dish (AB₃+CD₃+EF₃) /3 = R₃

Average mycelial growth of each strain is (R₁+ R₂ + R₃) /3 = F

Therefore, average mycelial growth of each species is (F₁+ F₂+…+F_n) / n, where n is number of strains used per species.

Effect of pH. A 5 mm diameter agar plug of an inoculum was removed with cork borer from 10 days old cultures grown on PDA and placed on the centre of each agar plate. The medium was adjusted to pH 5, 6, 7, 8 and 9 with the addition of 1N NaOH or 1N HCl before autoclaving and incubated for 10 days at 25 ºC. The measurement of mycelial growth was performed following the same technique described earlier.

Screening of favorable culture media. Ten different culture media namely Czapek Dox, Hamada, Hennerberg, Hoppkins, Glucose peptone, Glucose tryptone, Lilly, Mushroom complete, PDA and YM were prepared to investigate the mycelial growth of the studied mushrooms (Table 2). The media were adjusted to pH 6 before autoclaving. After autoclaving for 15 minutes at 121 ºC, 20~25 ml of each medium was aseptically poured into plates. A 5 mm diameter plug of an inoculum was removed from 10 days old culture grown on PDA and placed in the centre of each plate of 10 different culture media. After 10 days of incubation at 25 ºC, measurement of mycelial growth and density was performed as described above.

Table 2. Different media and their compositions used in this study.

*Media: Czapek Dox (Cza), Hamada (Ham), Hennerberg (Hen), Hoppkins (Hop), Glucose peptone (GP),
 Glucose tryptone (GT), Lilly (Lil), Mushroom complete (MC), Potato dextrose agar (PDA), Yeast-malt extract (YM).

Effect of carbon and nitrogen. To screen carbon and nitrogen sources favorable for the mycelial growth of selected mushrooms, the tests were performed on the basal medium [23] supplemented separately with each of 10 car-bon and 10 nitrogen sources. The basal medium was composed of: MgSO₄ - 0.05 g , KH₂PO₄ - 0.46 g , K₂HPO₄ - 1.0 g , thiamine-HCl - 120 μg, agar - 20 g and distilled water - 1000 ml. To screen carbon sources favorable for the mycelial growth, each carbon source together with 5 g of peptone was added separately to the basal medium at the final concentration of 0.1 M and mixed thoroughly [20]. The basal medium, which was used for screening of favorable nitrogen sources, was made of the same additives as those described by Sung et al. [23]._x Each nitrogen source together with 20 g of glucose was added to the basal medium at the final concentration of 0.02 M [20]. In both cases, the basal medium was adjustted to pH 6 before autoclaving for 15 minutes at 121 ºC and poured into plates. To measure colony diameter on the media, all plates were incubated for 10 days at 25 ºC._x Radial growth and density of mycelia were measured as already described.

Results and Discussion

Effect of temperature. The highest mycelial growth of tested mushrooms was obtained at 25 ºC while the lowest mycelial growth was recorded at 35 ºC. The fungi L. lepideus and P. ostreatus like high temperature (30 ºC). The lowest mycelial growth of these fungi appeared at 15 ºC (Figure 2). Lee et al., 1999, and Shim et al., 2003, reported that the mycelial growth of Paecilomyces fumosoroseus had been expedited gradually in proportion to the rise of temperature and the most suitable temperature was 25 ºC [12, 21]. Even though the mycelial growth of P. fumosoroseus was favored in the range of 20 to 25 ºC and had been expedited in proportion to the rise of temperature, the mycelial growth appeared to be suppressed at temperatures higher than 30 ºC. Imtiaj et al., 2007, reported that the temperature of 25 ºC was the most suitable for the majority of mushrooms for their spontaneous mycelial growth [11]. Sung et al., 1999, and Shim et al., 2005, stated that the mycelial growth of Macrolepiota procera and P. ostreatus was favored at 30 ºC, which is in accordance with our results [22, 24].

Effect of pH. To screen pH values favoring growth of mushrooms,thepH range of 5~9 was observed and the best pH was found to be 6~7. In case of L. lepideus and P. adiposa, the highest growth appeared at pH 5. The rest of the mushrooms chose pH 6~7 for their best mycelial growth (Figure 3). Shim et al. (2005) revealed that pH 7 was the most suitable for the optimal growth of M. procera [22]. Chi et al. (1996) and Choi et al. (1999) reported that mycelial growth of Phellinus japonica and P. linteus was optimal at pH 7 and 6~7, respectively [7, 8]. Shim et al. (2003) showed that optimal pH of Paecilomyces sinclairii was 8 [21]. Shim et al. (1997) also reported that the most favorable and most unfavorable pH of Grifola umbellata was 4 and 9, respectively [20]. This result suggested that mushrooms may have a broad pH range for their optimal mycelial growth.

Screening of favorable culture media. Ten different culture media were used to screen the optimal mycelial growth of different mushrooms. According to mycelial growth and density, Hamada, Lilly, PDA and YM were the most suitable while Czapek Dox, Hoppkins and Hennerberg were the most unfavorable media for the mycelial growth of mushrooms (Figure 4). Besides of slow growth, mycelial density was also somewhat thin to thin on Czapek Dox and Hoppkins media. This result corresponded to that obtained with P. sinclairii and P. fumosoroseus, which had been reported by Shim et al. (2003), where mycelial growth was optimal on Hamada medium [21]. Shim et al. (2005) also reported that PDA, YM, Mushroom complete and Hamada were the most suitable media, whereas Czapex Dox and Glucose peptone were unfavorable to mycelial growth of M. procera [22]. Adejoye et al. (2006) also reported that the Yeast and Malt extract medium enhanced the mycelial growth of Pleurotus florida [1].The mycelial growth in Hennerberg was slightly slo-wer growth than that in PDA and Hamada. It may be due to the ineffective utilization of microelements such as Mg⁺², SO₄^-2, NO₃^-1and PO₄^-3 by the mushrooms.

Effect of carbon sources. Ten different carbon sources were used to screen the optimal culture conditions. Among them, dextrin, fructose and sucrose were the best carbon sources for the mycelial growth. Lactose and galactose were the most unfavorable carbon sources. Maltose and mannose were also effective for the mycelial growth of studied mushrooms. In carbon sources, mycelial density (data not shown) varied from compact to somewhat compact (Figure 5). This result is completely similar to [22] and [20] but partially similar to [21]. Shim et al. (2005) proved that maltose, dextrin, sucrose and mannose were effective where lactose was highly ineffective [22]. Shim et al. (1997) reported that growth of G. umbellata was favored by the most of carbon sources except salicin, cellobiose and lactose^.[20]. Shim et al. (2003) revealed that dextrin was suitable for mycelial growth of P. fumosoroseus, which is similar to our findings, but they showed that mycelial density is thin in all carbon sources wheras our result is opposite [21]. Morrison and Boyd (1992) studied the utilization of carbon sources and explained that dextrin, which is a polymer of glucose, can be transformed to glucose during metabolic processes [15]. Garraway and Evans (1984) also found that glucose is the second best carbohydrate, which may be metabolized easily to produce cellular energy [9].

Effect of nitrogen sources. It was observed thatfor mycelial growth of mushrooms on the culture media the most suitable nitrogen sources were glycine, ammonium acetate and calcium nitrate while the most unsuitable were histidine and ammonium phosphate. Nitrogen sources stimulate the growth of mycelia where no mycelial growth was found on the culture media. In glycine supplemented medium, mycelial density was compact to somewhat compact while in the rest of nitrogen sources somewhat thin to thin mycelial density was observed (Figure 6). Imtiaj et al. (2007) studied the growth conditions of mushrooms and found that ammonium acetate and glycine strongly enhanced the growth of mushroom mycelia whereas ammonium phosphate and histidine discouraged the growth of mycelia [11]. Shim et al. (2005) clarified that glycine was the most favorable and histidine, arginine and ammonium oxalate were the most unfavorable for the mycelial growth of M. procera on the culture media [22]. Lee and Han (2005) showed that soytone, malt extract, yeast extract and bactopeptone were the most favorable while NaNO₃ and urea were the most unfavorable for the mycelial growth of Ramaria botrytis [13]. Moreover, organic nitrogen is more effective than inorganic nitrogen. The result of the present study is similar to the findings of Lee and Han [13].

Conclusion

This is the first study investigating the optimal conditions for mycelial growth of 371 strains belonging to 9 species of mushrooms. To obtain accurate growth conditions, a large number of mushroom strains of a given species were studied and it was found that the nutriational requirements for the mycelial growth of taxonomically identical strains were about similar. Thus, the basic information obtained from this study can be used for the mass production of mycelia of tested mushroom species.

Acknowledgement. This work was jointly supported by research grant from Korean Science and Engineering Foundation (KOSEF) and Agricultural R & D Promotion Center (ARPC) through Culture Collection of Wild Mushroom (CCWM) species in the University of Incheon, Korea.

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