The Effects of Temperature and Nutritional Conditions on Mycelium Growth of Two Oyster Mushrooms (Pleurotus ostreatus and Pleurotus cystidiosus) (2024)

Experimental design

The experiments were conducted in Plant Physiology and Value Added Microorganisms Laboratory, Department of Plant Industry, National Pingtung University of science and Technology (NPUST) in Taiwan during summer 2014 (April to July). The experiments were arranged in a randomized complete design with five replications per treatment.

Sources of materials

Two species of oyster mushroom PC (strain AG 2041) and PO (train AG 2042) were obtained from Plant Physiology and Value Added Microorganisms Laboratory, Department of Plant Industry, NPUST in Taiwan and maintained in potato dextrose agar medium (PDA). The different grains (brown rice, millet, yellow corn, wheat grain), materials to make culture media (yam, sweet potato, potato), lignocellulose substrates (sugarcane residue, acasia sawdust, mixed sawdust (sawdust made from mango and wax apple), corn straw, corn cob, cow manure, wild grass) were obtained from the different sources such as local farmers, local market, the campus of NPUST, bought from merchant in Pingtung county, except the coconut shell fiber was imported from Vietnam. The different chemical and instruments used were obtained from different companies in Taiwan and some were imported from Japan, United States, and Germany.

Temperature and nutritional conditions affecting the growth of mycelium

The effects of culture media, temperature, carbon sources and concentration, nitrogen sources and concentration, grain sources, lignocellulosic substrate on the mycelium growth of two species of oyster mushroom PO and PC for spawn production were studied.

Several trials were designed to evaluate factors affecting mycelium growth of two oyster mushroom species. For each test, mycelium discs with similar diameter (1 cm) were used. The discs were obtained by using the sterilized punch-hole tool to cut the mycelium and place into a new sterilized medium plate by using a transplant needle. The diameter of the mycelium extension was measured every 2 days with the help of a transparent ruler at the same time (between 9:00 AM and 12:00 PM) and surface mycelium density was observed.

Statistical analysis

One-way analysis of variance (ANOVA) was conducted with Duncan's multiple range tests to compare the mean significant differences (p < 0.05) among treatments by using computer software SAS Version 9.1 (SAS Institute Inc., Cary, NC, USA). Each value is expressed as mean ± SE (n = 5).

Effect of different culture media on the mycelium growth

Every living being requires food for its growth and reproduction and oyster mushrooms are not an exception to it. In order to culture the oyster mushroom in the laboratory, it is necessary to furnish those compounds in the media which are requires for its growth and other life process. In the present study, two oyster mushrooms PO and PC were cultured successfully in the laboratory environment. During the present investigation, different media, i.e., PDA, MEA, SPDA, YDA were used to identify their effect on mycelium growth. Data (Table 1) showed that the observed mycelium colony diameter of mushroom PO on four different media significantly differed at (p < 0.05) while the mycelium colony diameter of mushroom PC on four media did not significantly differ. Mycelium densities of both oyster mushroom PO and PC were compact in all media. The average mycelium diameter of two oyster mushroom species ranged from 3.92 ± 0.05 to 9.00 ± 0.00 cm at 8 days after inoculation (DAI). The mycelium growth of mushroom PO on PDA, YDA media was better than those on MEA, SPDA media at 2, 4, and 6 DAI; however, they were not significantly different at 8 DAI. The result indicated that these four cultural media are suitable for mycelium growth of mushroom PC while PDA and YDA media are more suitable for the mycelium growth of mushroom PO. This may be due to availability of required nutrients for mushroom PO in PDA and YDA media.

The results of the present research showed that the mycelium of PO took shorter time, but in the case of PC, the mycelium took longer time to complete growth in all media (data not shown). These findings were similar to the results reported by Mshandete and Mgonja [13]; Mansur et al. [14]. They indicated that PDA and YDA were suitable media for culture of Pleurotus species to achieve high level mycelium biomass, exopolysacharides and mycelium protein. In Taiwan, potato is so much cheaper than yam, sweet potato, so PDA medium was more suitable and efficient to use as alternatives for culture of mushroom PO and PC. From those results, we chose local materials as cheaper materials to prepare media for culture of mushroom PO and PC.

Effect of temperature on mycelium growth

Temperature is a very important environment factor for mycelium growth of fungi. To determine optimal temperature for mycelium growth, two species of oyster mushroom were cultivated in PDA medium at various temperatures (16℃, 20℃, 24℃, 28℃, 32℃, and 36℃). The trend of mycelium growth in response to temperature was very similar for the two oyster mushroom species, where the optimum temperature for both oyster mushroom species was found to be 28℃ followed by 32℃ and 24℃ (Table 2, Fig. 1). The mycelium growth of mushroom PO was significantly faster than that of mushroom PC at each temperature tested and PC did not seem to grow after 8 days inoculation at 36℃. The mycelium density of mushroom PC was very thin at 16℃ and 36℃. Neelam et al. [15] indicated that the optimum temperature for mycelium growth of oyster mushroom P. florida was 25~30℃. This optimum temperature result indicated that the two species of oyster mushroom PO and PC were able to grow better in summer and autumn season in subtropical and tropical regions as potential opportunity to develop oyster mushroom production in poor and developing countries in Asia. The result is quite similar to the results reported by Kashangura [16], Choi et al. [17] when demonstrating that the mycelium growth and fruiting formation of oyster mushroom species were affected by temperature and they could grow at high temperature as summer season in tropical regions.

Effect of carbon sources and sucrose concentration on mycelium growth

Carbohydrates, which play key roles as structural and storage compounds in cell, are distinguished as monosacharides, disacharides, and polysacharides. Mycelium of many mushrooms will grow to some extent over a wide range of carbon sources. In order to find the optimal carbon, different kinds of carbon sources (2% concentration) including glucose, dextrose, fructose, maltose, sucrose, and molasses were added into the basal medium (PA medium). The result shown in Table 3 reveals the suitability of various carbon sources for mycelium growth of oyster mushroom PO and PC. The result indicated that there was a significant difference in mycelium growth of mushroom PO and PC grown on various carbon sources. Among seven carbon sources tested, glucose, sucrose, and molasses were favorable to the mycelium growth (mycelium colony diameter and mycelium density) of oyster mushroom PO, while glucose, sucrose, and dextrose also gave good mycelium growth of oyster mushroom PC (Table 3). Six DAI, the highest mycelium colony diameters of oyster mushroom PO were obtained from the media containing glucose (8.60 ± 0.10 cm), sucrose (8.54 ± 0.12 cm), and molasses (8.34 ± 0.09 cm), while the highest mycelium colony diameters of mushroom PC were recorded from the media containing glucose (3.50 ± 0.09 cm), sucrose (3.54 ± 0.02 cm), and dextrose (3.44 ± 0.05 cm). On the contrary, six DAI, the slowest mycelium growth obtained in the medium containing lactose as carbon source for both oyster mushroom PO and PC was 3.36 ± 0.10 cm and 2.50 ± 0.07 cm. Fu et al. [18], reported that sucrose was the best carbon source for mycelium growth of fungus Villosiclava virens. Neelam et al. [15] also indicated dextrose was the best carbon source for mycelium growth of P. florida. Mao et al. [19] demonstrated that glucose was an optimal carbon source for cordycepin production of Chinese traditional medicinal mushroom Cordyceps militaris. Moreover, glucose was also identified as the best carbon source for exopolysaccharides production of edible mushrooms such as Phellinus [20]. The preference of glucose, sucrose, and molasses by oyster mushroom PO and glucose, sucrose, and dextrose by oyster mushroom PC were may be due to the ease with which it was metabolized to produce cellular energy for the growth of organism. Kurtzman and Zadražil [21] mentioned that the ability of Pleurotus species to use different carbon sources may be an expression of the physiological differences in the species or of the isolates, since other isolates of the same species might give different results.

Among good carbon sources (glucose, dextrose, sucrose, and molasses) for PO and PC, sucrose and molasses are the cheapest sources. Sucrose gave significantly highest mycelium colony diameter for both oyster mushrooms PO and PC, while molasses only gave significantly highest mycelium growth of oyster mushroom PO. Sucrose also gave the compact mycelium density of both oyster mushroom PO and PC. Hence, sucrose was more suitable and effective than others for large scale production of oyster mushroom PO and PC. It was chosen as carbon source in the following test.

Among sucrose concentrations 1~5% (10~50 g/L), there was no significant difference in mycelium colony diameter of oyster mushroom PO (Fig. 2). However, six DAI, sucrose concentrations from 1% to 5% gave the highest mycelium colony diameter (8.28~8.62 cm) and significantly higher than those of PA media (control) and other sucrose concentrations. The highest mycelium colony diameter of mushroom PC (3.22~3.44 cm) was also achieved at 1~3% concentration levels of sucrose. However, high sucrose concentrations (more than 3% for PC and more than 5% for PO) were unable to increase mycelium colony diameter. This was explained that the osmotic pressure caused by a high sucrose concentration may be detrimental to the metabolite biosynthesis. On the other hand, a too high carbon concentration also made too high carbon/nitrogen ratio, which will inhibit the mycelium growth. Bai et al. [22] indicated that the low carbon concentration (30 g glucose/L) gave the highest mycelium growth and yield of betulin of Inonotus obliquus when compared to 40 and 50 g glucose/L. A high initial glucose concentration (55 or 70 g/L) was unfavourable to cordycepin biosynthesis of medicinal mushroom Cordyceps militaris [19].

Effect of nitrogen sources and concentration on mycelium growth

Nitrogen is an essential element required by all fungi for the synthesis of nitrogen containing compounds such as purines, pyrimidines, protein and for the cell wall component chitin, which is composed of β(1-4)-linked unit of N-acetylglucosamine [23]. The chemical composition of culture medium, especially the nitrogen concentration, is a mean of physiological control and regulation of microorganism metabolism [24]. In this research, both organic nitrogen and inorganic nitrogen sources were used to evaluate their effects on mycelium growth of oyster mushroom PO and PC.

There was a significant difference in mycelium growth of PO and PC among nitrogen sources (Table 4, Fig. 3). Six DAI, the significantly highest mycelium colony diameters of PO and PC (8.84 ± 0.07 cm and 3.70 ± 0.05 cm, respectively) were obtained in media containing ammonium chloride (NH₄Cl) as nitrogen source. Ammonium sulfate (N₂H₈SO₄) is the second best nitrogen source for mycelium growth of both PO and PC but the mycelium colony diameter of oyster mushroom PO was not significantly different as compared to control medium. Six DAI, the significantly lowest values of mycelium colony diameter of PO and PC (5.06 ± 0.06 cm and 3.16 ± 0.06 cm, respectively) were obtained in medium containing soybean (PO) and urea (PC) (Table 4, Fig. 3). There was an increase of mycelium density in all treatments added different nitrogen sources (except urea). However, mycelium colony diameters of both mushroom PO and PC were not improved in media containing some nitrogen sources such as malt extract, soybean, and yeast extract. Mycelium density occurred in the medium containing organic nitrogen was more compact than that in the medium using inorganic nitrogen except for the medium containing ammonium chloride (Table 4, Fig. 3). In another study, Cheng et al. [25] demonstrated that ammonium chloride was one of the nitrogen sources to get high dry weight of the fungus Cryphonectria parasitica. Neelam et al. [15] also reported that ammonium chloride supported growth of mycelium of P. florida and P. ostreatus better than sodium nitrate and calcium nitrate because nitrate ions have been implicated in the inhibitory effect of some basidiomycetes, which may be difficult to transport across the fungal membrane where it can promote growth. The result is in agreement with Fu et al. [18] who showed that ammonium chloride and ammonium sulfate were the most suitable nitrogen sources for mycelium growth of Villosiclava virens and in comparison with nitrate nitrogen sources, the ammonium nitrogen sources were favorable for mycelium growth.

Based on the above results, ammonium chloride was selected as a suitable nitrogen source for further studies. Fig. 4 summarized the effects of initial ammonium chloride concentrations on the mycelium growth of oyster mushroom PO and PC. The data presented in Fig. 4 indicate that the mycelium growth of oyster mushroom PO increased with the increasing of ammonium chloride concentration from 0.01% to 0.09% in PSA medium. The mycelium growth of oyster mushroom PC also increased with the concentration of ammonium chloride from 0.01% to 0.05%. The highest colony diameter of PO mycelium achieved with the ammonium chloride concentrations from 0.03% to 0.09%, while the PC mycelium achieved the highest colony diameter with the ammonium chloride concentrations from 0.03% to 0.05%. The mycelium growth of PO and PC decreased when the ammonium chloride concentrations are over 0.09% and 0.05%, respectively. It may be due to a high concentration of nitrogen source leading to a too low carbon/nitrogen ratio, which inhibited the growth of mycelium. In a study using different nitrogen rich supplements for P. florida cultivation on corn cob substrate, Naraian et al. [26] demonstrated that the low concentration (0.5%) of ammonium sulfate and urea is better for mycelium growth rate of P. florida than the higher concentration (1% and 1.5%). On the other hand, in another study, oyster mushroom yield decreases when the ammonia concentration is higher than 68 ppm [27].

Effect of different grain sources on the mycelium growth

Grains which are rich in protein and carbohydrate, usually used as media to produce spawn of edible mushrooms. In the present research, four different grains including brown rice, yellow corn, wheat, and millet were used to indicate their effects on spawn production of oyster mushroom PO and PC (Table 5). The result indicated that, in all grain cases, mycelium extensions of mushroom PC and PO were significantly slower than that of control treatments (Table 5). When compared to mycelium extension of mushroom PC, mycelium extension of mushroom PO was higher. Mycelium densities of both oyster mushroom PO and PC on grain media were compact and somewhat compact. Brown rice was found to be the most favorable to the mycelium growth of mushroom PO and PC. On the 10th day of incubation, the highest mycelium colony diameters of PO and PC obtained in brown rice medium were 8.88 ± 0.12 cm and 3.96 ± 0.17 cm, respectively. Yellow corn and wheat were next to brown rice with mean values for mycelium colony diameter of PO being 8.28 ± 0.12 cm and 8.10 ± 0.11 cm, respectively. Those values of PC were 3.28 ± 0.12 cm and 3.32 ± 0.15 cm, respectively. All grains supported the mycelium growth of two studied Pleurotus species but in varying degrees. The least support for both species studied was shown by millet. This may be due to millet grain size being very small when compared to brown rice, wheat, yellow corn. Tinoco et al. [28] found that larger surface and pore of substrates were better supports for mycelium growth rate. This could account for the results recorded by brown rice, wheat and yellow corn. Sofi et al. [29] reported that mycelium colony diameter of P. ostreatus fungi MTCC-1801 in various grains was significantly affected by substrate style. The author also indicated that the larger surface area and spore of substrates are responsible for the more mycelium growth rate.

Effect of different lignocellulosic substrate sources

In this research, some agroforestry wastes such as sugarcane residue, acasia sawdust, mixed sawdust, corn straw, corn cob, cow manure, wild grass, and coconut shell fiber were used as substrates to evaluate their effect on the mycelium growth of oyster mushroom PO and PC. The data summarized in Table 6 indicated that the mycelium growth of oyster mushroom PO and PC in all cases was significantly lower than that in control media. The mycelium growth trend of two oyster mushroom species in response to lignocellulosic substrates was very similar. Ten DAI, the greatest values of mycelium colony diameter of oyster mushroom PO and PC were recorded at sugarcane residue substrate (6.68 ± 0.05 cm and 3.66 ± 0.10 cm, respectively). However, they were not significantly different when compared to those in acasia sawdust substrate. The lowest mycelium colony diameters were obtained in coconut shell fiber for both PO and PC mushroom. Table 6 showed that substrates made from sugarcane residue, acasia sawdust, and corn cob were more suitable for the mycelium growth (including mycelium colony diameter and density) of both mushroom PO and PC as compared to mixed sawdust, corn straw, cow manure, wild grass and coconut shell fiber. This may be due to rich carbon and nitrogen composition in these substrates which support good mycelium growth. Moreover, the saprophytic ability of those fungi also supported to produce extracellulase hydrolyzing enzymes (cellulose, laccase, and lignase) to hydrolyze cellulose, lignin and hemicelluloses present in these substrates to simple sugars for growth. On the other hand, Badu et al. [30] demonstrated that the nutrient content, the growth and yield of oyster mushroom cultivated on sawdust depend on the chemical constituents such as the cellulose content, the hemicelluloses content and the lignin content. Substrates made from Triplochiton scleraxylon containing high concentration of lignin (34.08%) gave the lower yield of mushroom than that in Ceiba pentandra sawdust substrates containing low rate of lignin (27.55%). Shah et al. [31] reported that sawdust gave maximum yield and the leaves gave minimum yield of PO. In another study, Kuforiji and Fasidi [32] also reported that substrates such as corn cob, rice straw, and sawdust of Mansonia altissima and Boscia angustifolia supported the growth of Pleurotus tube-regiun.

Based on the above results, sugarcane residue, acasia sawdust, and corn cob were selected as the most suitable lignicellulosic substrate sources to produce PO and PC mushroom spawn besides spawn source produced from grains. Moreover, these lignocellulosic substrates were also suggested to cultivate oyster mushroom PO and PC.

In conclusion, the mycelium growth of oyster mushroom PO and PC were affected by different temperature conditions and nutritional sources. It was concluded that PDA, SPDA, YDA, and MEA were the most favourable media for the mycelium growth of mushroom PC while PDA and YDA were the most suitable media for mycelium growth of oyster mushroom PO. Maximum mycelium growth of oyster mushroom PO and PC was achieved by cultivating them at the optimum temperature of 28℃. In regarding to carbon source and concentration, PO mushroom was identified to be the most suitable to PA medium with 1~ 5% sucrose concentration, while 1~3% sucrose concentration gave the good mycelium growth of oyster mushroom PC. This study also determined that ammonium chloride concentrations at 0.03~0.09% and 0.03~0.05% gave the greatest values in mycelium colony diameter of oyster mushroom PO and PC, respectively. Regarding to grain and lignicellulosic substrate sources to produce mushroom spawn, brown rice followed by yellow corn and wheat was found to be favourable grains, while sugarcane residue, acasia sawdust, and corn cob were suitable lignicellulosic substrate sources for the mycelium growth of oyster mushroom PO and PC. In all studied cases, mycelium growth of oyster mushroom PO was significantly better than that of oyster mushroom PC.

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