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- Saudi J Biol Sci
- v.20(4); 2013 Oct
- PMC3824138
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Saudi J Biol Sci. 2013 Oct; 20(4): 333–338.
PMCID: PMC3824138
PMID: 24235869
WenJie Yang,a,⁎ FengLing Guo,b and ZhengJie Wana
Author information Article notes Copyright and License information PMC Disclaimer
Abstract
Oyster mushroom (Pleurotus ostreatus) was cultivated on rice straw basal substrate, wheat straw basal substrate, cotton seed hull basal substrate, and wheat straw or rice straw supplemented with different proportions (15%, 30%, and 45% in rice straw substrate, 20%, 30%, and 40% in wheat straw substrate) of cotton seed hull to find a cost effective substrate. The effect of autoclaved sterilized and non-sterilized substrate on growth and yield of oyster mushroom was also examined. Results indicated that for both sterilized substrate and non-sterilized substrate, oyster mushroom on rice straw and wheat basal substrate have faster mycelial growth rate, comparatively poor surface mycelial density, shorter total colonization period and days from bag opening to primordia formation, lower yield and biological efficiency, lower mushroom weight, longer stipe length and smaller cap diameter than that on cotton seed hull basal substrate. The addition of cotton seed hull to rice straw and wheat straw substrate slowed spawn running, primordial development and fruit body formation. However, increasing the amount of cotton seed hull can increase the uniformity and white of mycelium, yield and biological efficiency, and increase mushroom weight, enlarge cap diameter and shorten stipe length. Compared to the sterilized substrate, the non-sterilized substrate had comparatively higher mycelial growth rate, shorter total colonization period and days from bag opening to primordia formation. However, the non-sterilized substrate did not gave significantly higher mushroom yield and biological efficiency than the sterilized substrate, but some undesirable characteristics, i.e. smaller mushroom cap diameter and relatively long stipe length.
Keywords: Cotton seed hull, Non-sterilized substrate, Pleurotus ostreatus, Rice straw, Sterilized substrate, Wheat straw
1. Introduction
Cultivation of edible mushrooms might be the only current process that combines the production of protein-rich food with the reduction of environmental pollution (Sánchez, 2010). It represents one of the most efficient biotechnological processes for lignocellulosic organic waste recycling (Mandeel et al., 2005). Oyster mushroom has been widely cultivated in many different parts of the world. It has abilities to grow at a wide range of temperatures utilizing various lignocelluloses (Sánchez, 2010). In China, oyster mushrooms are cultivated mainly on sawdust and cotton seed hull. Demand for sawdust and cotton seed hull is increasing following the increasing expansion in the poultry industry and mushroom cultivation, thus making it difficult and expensive for commercial mushroom growers to get sawdust and cotton seed hull.
Large volumes of rice and wheat straw are produced as agricultural by-products. Currently, wheat straw and/or rice straw are disposed off through open-field burning, which leads to serious environment pollution problem. If rice straw and/or wheat straw can support the growth of oyster mushroom, then it would be one of the solutions to transform these inedible wastes into an accepted edible biomass of high market value, and serve as a cheap source of substrate for mushroom grower. However, the utilization of rice straw and/or wheat straw in oyster mushroom cultivation is not popular in China for its low yield and low biological efficiency (Wang, 2010; Fan et al., 2011; Li et al., 2011), or the need for varying degrees of pre-treatment (Li et al., 2002, 2011). Thus, the present work was carried out with objectives of using rice straw and/or wheat straw as a basal substrate supplemented with various ratios of cotton seed hulls to produce oyster mushrooms, and evaluate the effect of supplement levels in substrate sterilized or not on mushroom yield and size of oyster mushroom.
2. Materials and methods
2.1. Microorganism and spawn preparation
Pleurotus ostreatus, obtained from the Mushroom Spawn Research Center of Huazhong Agricultural University (Wuhan City, Hubei, China), was grown on the potato dextrose agar (PDA 200g/l diced potatoes; 20g/l glucose; 15g/l agar) medium at 25°C for regular subculture. Oyster mushroom spawn was prepared in 850-ml polypropylene plastic bottles filled with cotton seed hull 87%, wheat bran 10%, sucrose 1%, plaster stone 1%, and calcium superphosphate 1% (w/w, in terms of dry weight), which were widely used in central China, and then autoclave sterilized at 121°C for 80min. After cooling down to room temperature, the sterilized cotton seed hulls of every bottle were inoculated with 5cm2 mycelial agar discs. The spawn was incubated in the laboratory at 26±2°C and 70% relative humidity for two weeks.
2.2. Substrate preparation, inoculation, and incubation
The straws were completely dried under the Sun, and then chopped into 4cm lengths, and soaked in water for overnight before substrate preparation. After draining the excess water, they were used as substrate for replacing partially wheat bran and cotton seed hull. In order to determine suitable substrates and suitable ratios for the cultivation of oyster mushroom, various materials and combination substrates were tested (Table 1).
Table 1
Materials used for substrate preparation and their mixture ratios and analysis (C/N ratio).
| Substrate and mixture ratio by weight | Symbol | C/N ratio |
|---|---|---|
| Cotton seed hull 80%+wheat bran 20% | C80+WB20 | 34.87 |
| Rice straw 80%+wheat bran 20% | R80+WB20 | 49.19 |
| Wheat straw 80%+wheat bran 20% | WS80+WB20 | 64.63 |
| Cotton seed hull 45%+Rice straw 45%+wheat bran 10% | C45+R45+WB10 | 44.76 |
| Cotton seed hull 30%+Rice straw 60%+wheat bran 10% | C30+R60+WB10 | 47.45 |
| Cotton seed hull 15%+Rice straw 75%+wheat bran 10% | C15+R75+WB10 | 50.13 |
| Cotton seed hull 40%+wheat straw 40%+wheat bran 20% | C40+WS40+WB20 | 49.75 |
| Cotton seed hull 30%+wheat straw 50%+wheat bran 20% | C30+WS50+WB20 | 53.47 |
| Cotton seed hull 20%+wheat straw 60%+wheat bran 20% | C20+WS60+WB20 | 57.19 |
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Water content of the final mixture was adjusted to 65% (w/w). The sterilized substrate cultivation was that the mixture was filled into polyethylene bags and autoclave sterilized at 121°C for 80min. After the substrates were cooled down to room temperature, they were inoculated with 5g of oyster mushroom spawn. The non-sterilized substrate cultivation was that three layers of 5g of oyster mushroom spawn per layer were inoculated in the bottom, middle and surface of substrate when the mixture was filled into polyethylene bags. Twenty replicate polyethylene bags were used for each substrate.
The inoculated substrates were kept in a spawn running room at 25°C and 70% relative humidity under dark conditions. The spawn run period to total colonization (the number of days from inoculation to complete colonization of the substrate by the mycelium) was recorded. The mycelial growth rate was determined as the height (mm) of mycelia in the colonized culture bag divided by the incubation time (days).
2.3. Cropping, harvest and determination of biological efficiency
After the primordia appeared on the top layer of substrate in each polyethylene bag, the bag was moved to a cropping room in which the temperature was controlled at 28°C, relative humidity at 80% or above, and light intensity at about 100lux. The cropping room was watered intermittently to maintain the moisture during the cropping time.
Mushrooms were harvested when the mushroom cap surface were flat to slightly up-rolled at the cap margins. The harvested fruiting bodies in each bag were then counted and weighed. At the end of the harvest period, the accumulated data were used to calculate the biological efficiency and mushroom weight.
Biological efficiency (%)=eight of fresh mushrooms harvested per bag/weight of dry substrate per bag×100.
Mushroom weight (g)=Total weight of harvested fresh mushrooms per bag/total number of mushrooms harvested per bag.
2.4. Substrate analysis
Tested samples of substrate after sterilization were dried at 60°C to a constant weight, then ground into a coarse powder (8openings/cm) using a mill. The method used to obtain carbon content was that of Nelson and Sommers (1982) and nitrogen content was measured using the Kjeldahl method. Then the carbon/nitrogen ratio of each substrate was calculated.
2.5. Statistical analysis
Differences between the means of individual groups were assessed by one-way ANOVA with Duncan’s multiple range tests.
3. Results
3.1. Chemical composition and colonization of different substrates
C/N ratio varied considerably among substrates (Table 1). The mycelial growth rate, surface mycelial density, total colonization period and days from bag opening to primordia formation of oyster mushroom cultivation on different substrates are presented in Table 2.
Table 2
Mycelial growth rate (mm/day), surface mycelial density, total colonization period and days from bag opening to primordia formation of oyster mushroom cultivated in different substrates. Letters indicate means among substrates followed by the same letter are not statistically different (P>0.05).
| Substrate | Mycelial growth rate (mm/day) | Surface mycelial densitya | Total colonization period (days) | Days from bag opening to primordia formation (days) | ||||
|---|---|---|---|---|---|---|---|---|
| Sterilized | Non-sterilized | Sterilized | Non-sterilized | Sterilized | Non-sterilized | Sterilized | Non-sterilized | |
| C80+WB20 | 6.45d⁎ | 10.00c | +++ | ++ | 31a⁎ | 21a | 21a⁎ | 14a |
| R80+WB20 | 12.50ab⁎ | 14.26a | ++ | + | 16c | 15bc | 10c⁎ | 6bc |
| C45+R45+WB10 | 10.53c⁎ | 13.33b | +++ | ++ | 22b | 18b | 15b⁎ | 9b |
| C30+R60+WB10 | 11.11bc⁎ | 14.16a | +++ | ++ | 18c | 16bc | 14b⁎ | 8b |
| C15+R75+WB10 | 11.76bc⁎ | 14.21a | ++ | ++ | 17c | 14c | 10c | 8b |
| WS80+WB20 | 13.33a⁎ | 14.29a | ++ | + | 15c | 14c | 9cd⁎ | 3c |
| C40+WS40+WB20 | 11.11bc⁎ | 13.33b | +++ | ++ | 21b | 18b | 14b⁎ | 9b |
| C30+WS50+WB20 | 11.76bc⁎ | 14.11ab | +++ | ++ | 17c | 15bc | 14b⁎ | 8b |
| C20+WS60+WB20 | 12.50ab⁎ | 14.17a | ++ | ++ | 16c | 14c | 7d⁎ | 4c |
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aDegree of mycelial density when the mycelia fully colonizes the substrate: + poor running growth, ++ mycelium grows throughout the whole bag but is not uniformly white, +++ mycelium grows throughout the whole bag and is uniformly white.
⁎Indicates means within a substrate followed by the same letter are statistically different (P<0.05).
The mycelial growth on substrate W80+WB20 and R80+WB20 gave significantly faster mycelial growth rate than that on substrate C80+WB20 for both sterilized and non-sterilized substrate. For both sterilized and non-sterilized substrates, the mycelial growth on substrates C45+R45+WB10 was significantly slower than that on substrate R80+WB20, and that on the substrates C40+WS40+WB20, C30+WS50+WB20 were significantly slower than that on substrate WS80+WB20. The results indicated that addition of cotton seed hull decreased mycelial growth compared to sole rice straw or wheat straw substrate. Compared to the sterilized substrate, the non-sterilized substrate exhibited significantly higher mycelial growth rate.
The surface mycelial density of the mycelium was comparatively poor on sterilized substrates R80+WB20, WS80+WB20, C15+R75+WB10 and C20+WS60+WB20. Surface mycelial density on substrates C80+WB20, C45+R45+WB10, C30+R60+WB10, C40+WS40+WB20 and C30+WS50+WB20 was uniform and white. Compared to the sterilized substrate, the surface mycelial density on non-sterilized substrate was comparatively poor.
The total colonization period on substrates WS80+WB20 and R80+WB20 was significantly shorter than that on substrate C80+WB20. Total colonization period on substrate R80+WB20 was significantly shorter than that on substrate C45+R45+WB10 for sterilized substrate, and that on substrate C40+WS40+WB20 was significantly longer than that on substrate WS80+WB20 for both sterilized and non-sterilized substrate. Addition of cotton seed hull could increase the total colonization period compared to sole rice straw or wheat straw substrate. Significant difference between sterilized substrate and non-sterilized substrate in terms of total colonization period occurred on substrate C80+WB20.
The days from bag opening to primordia formation ranged from 3 to 14days for non-sterilized substrate, 7 to 21days for sterilized substrate. Days from bag opening to primordia formation on substrates C45+R45+WB10 and C30+R60+WB10 were significantly longer than those on R80+WB20, and on substrate C40+WS40+WB20 were significantly longer than those on WS80+WB20. Addition of cotton seed hull significantly delayed primordia formation compared to sole rice straw or wheat straw substrate. Significant difference between sterilized substrate and non-sterilized substrate in terms of days from bag opening to primordia formation occurred on all substrate except C15+R75+WB10.
3.2. Mushroom production
Flushes of mushroom production varied among different substrates (Table 3). There were at least three flushes for all non-sterilized substrates during cultivation. There were 1–3 more flushes on non-sterilized substrate than those on sterilized substrate for most substrate. The maximum average yield of fruit bodies (>60%) was obtained in the first two flushes on all the substrates used under experimental conditions.
Table 3
Mean±standard deviation (SD) number of flushing mushroom, yields and biological efficiency (BE) on different substrates. Letters indicate means among substrate followed by the same letter are not statistically different (P>0.05).
| Substrate | Number of flushing | Yields of mushrooms (g) | Biological efficiency (BE %) | |||
|---|---|---|---|---|---|---|
| Sterilized | Non-sterilized | Sterilized | Non-sterilized | Sterilized | Non-sterilized | |
| C80+WB20 | 5±0.7 | 7±1.0 | 438.1±14.3a | 425.3±20.7a | 125.6±8.91a | 121.5±11.4a |
| R80+WB20 | 2±0.1 | 3±0.9 | 223.7±14.5e | 202.7±16.9d | 53.9±5.66d | 50.9±6.32c |
| C45+R45+WB10 | 4±0.6 | 5±0.7 | 339.4±15.6bc | 319.5±16.3b | 96.7±6.88b | 91.2±9.62b |
| C30+R60+WB10 | 4±0.6 | 5±0.8 | 324.2±9.8c | 312.6±12.9b | 89.6±5.54b | 72.3±9.78b |
| C15+R75+WB10 | 3±0.7 | 4±0.8 | 261.5±18.7d | 264.7±20.1c | 74.7±4.66c | 71.6±7.49b |
| WS80+WB20 | 3±0.8 | 3±0.8 | 214.6±17.8e | 190.1±26.4d | 51.3±4.79d | 44.3±6.54c |
| C40+WS40+WB20 | 4±0.9 | 5±1.0 | 348.7±21.2b | 309.9±24.5b | 94.6±4.55b | 88.5±9.64b |
| C30+WS50+WB20 | 3±0.5 | 5±1.0 | 318.3±17.7c | 312.1±20.2b | 90.9±5.46b | 86.1±8.73b |
| C20+WS60+WB20 | 3±1.0 | 4±0.8 | 268.1±16.3d | 268.5±19.8c | 76.6±5.85c | 74.7±9.18b |
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Oyster mushroom on substrate C80+WB20 has highest yield and biological efficiency, significantly higher than that on substrates R80+WB20 and WS80+WB20. Oyster mushroom on substrates C45+R45+WB10 and C30+R60+WB10 gave significantly higher yield and biological efficiency (BE) than that on substrate R80+WB20, and on substrates C40+WS40+WB20 and C30+WS50+WB20 gave a significantly higher yield and BE than that on substrate WS80+WB20. Addition of cotton seed hull significantly increased mushroom weight compared to sole rice straw or wheat straw substrate. The mushroom yield and biological efficiency on non-sterilized substrate was not significantly different from that on sterilized substrate, with much lower average yield per flush (Table 3).
3.3. Mushroom weight, cap diameter and stipe length
The mushroom weight, cap diameter and stipe length development in oyster mushroom were significantly affected by substrate types and substrate mixture as shown in Table 4.
Table 4
Mean mushroom weight (g), cap diameter (cm), stipe length (cm) of oyster mushroom (Pleurotus ostreatus) grown on different substrates. Letters indicate means among substrate followed by the same letter are not statistically different (P>0.05).
| Substrate | Mean mushroom weight (g) | Cap diameter (cm) | Stipe length (cm) | |||
|---|---|---|---|---|---|---|
| Sterilized | Non-sterilized | Sterilized | Non-sterilized | Sterilized | Non-sterilized | |
| C80+WB20 | 25.13a⁎ | 22.68a | 10.4a⁎ | 9.2a | 2.3d⁎ | 2.7c |
| R80+WB20 | 16.66d | 16.33c | 7.5c | 7.0c | 3.1ab | 3.2b |
| C45+R45+WB10 | 21.41b⁎ | 19.11b | 9.6ab⁎ | 8.6b | 2.5cd⁎ | 2.9c |
| C30+R60+WB10 | 19.85bc⁎ | 17.67bc | 8.6bc | 8.1b | 2.5cd⁎ | 2.9c |
| C15+R75+WB10 | 17.33cd | 16.62c | 7.6c | 7.0c | 3.1ab | 3.4ab |
| WS80+WB20 | 16.85d | 16.22c | 6.3d | 5.9d | 3.5a | 3.7a |
| C40+WS40+WB20 | 22.93b⁎ | 18.82b | 9.4ab⁎ | 8.2b | 2.6bc⁎ | 3.0bc |
| C30+WS50+WB20 | 19.19bc⁎ | 17.60bc | 8.8bc⁎ | 8.0b | 2.9b⁎ | 3.3b |
| C20+WS60+WB20 | 17.77cd | 16.94c | 7.8c | 7.1c | 3.2ab⁎ | 3.6a |
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⁎Indicates means within a substrate followed by the same letter are statistically different (P<0.05).
The oyster mushroom grown on substrates R80+WB20 and WS80+WB20 had significantly lower mushroom weight than that on substrate C80+WB20. The oyster mushroom grown on substrates C45+R45+WB10 and C30+R60+WB10 gave significantly higher mushroom weight than that on substrate R80+WB20 for sterilized substrate, and on substrate C45+R45+WB10 gave significantly higher mushroom weight than that on substrate R80+WB20 for non-sterilized substrate. Oyster mushroom grown on substrates C40+WS40+WB20 and C30+WS50+WB20 gave significantly higher mushroom weight than that on WS80+WB20 for sterilized substrate, and substrate C40+WS40+WB20 gave significantly higher mushroom weight than that on substrate WS80+WB20 for non-sterilized substrate. When compared to sterilized substrate, the mushroom on non-sterilized sterilized substrates C45+R45+WB10, C30+R60+WB10, C40+WS40+WB20, C30+WS50+WB20 and C80+WB20 has significantly larger mushroom weight.
The oyster mushroom grown on substrates R80+WB20 and WS80+WB20 gave significantly smaller cap diameter than that on substrate C80+WB20. Oyster mushroom grown on substrate C45+R45+WB10 had significantly larger cap diameter than that on substrate R80+WB20, and on substrate C40+WS40+WB20 had significantly larger cap diameter than that on substrate WS80+WB20. The oyster mushroom on sterilized substrates C45+R45+WB10, C30+R60+WB10, C40+WS40+WB20, C30+WS50+WB20 and C80+WB20 had significantly larger cap diameter compared to non-sterilized same substrate.
The oyster mushroom grown on substrates R80+WB20 and WS80+WB20 gave the significantly longer stipe length than that on substrate C80+WB20. The oyster mushroom grown on substrate C45+R45+WB10 gave significantly shorter stipe length than that on substrate R80+WB20, and grown on substrate C40+WS40+WB20 had significantly shorter stipe length than that on substrate WS80+WB20. When compared to non-sterilized substrate, the oyster mushroom on sterilized substrates C80+WB20, C45+R45+WB10, C30+R60+WB10, C40+WS40+WB20, C30+WS50+WB20 and C20+WS60+WB20 has significantly shorter stipe length.
Generally speaking, increasing the amount of cotton seed hull in the substrate mixture slowed spawn running, primordial development and fruit body formation. However, increasing amount of cotton seed hull increased the uniformity and white of mycelium, yield and BE, and increase mushroom weight, enlarged cap diameter and shorten stipe length of oyster mushroom (Tables 2–4).
4. Discussion
Among the nine substrates for the cultivation of oyster mushroom, wheat straw and rice straw gave the faster mycelial growth rate, time to primordial formation, time to first crop than cotton seed hull, however, this did not correspond with mushroom size and yield.
Oyster mushroom on rice straw and wheat straw substrate in the present study have much low yield and BE, similar to the results of Zhang et al. (2002), but lower than some reported cultivation which have relatively good yields (Obodai and Vowotor, 2003). The reason may be physical nature and high C/N ratio of rice straw and wheat straw that were not suitable for the cultivars of oyster mushroom used in China. Rice or wheat straw substrate in the present study was also found to be very susceptible to drying, also reported by He et al. (1995) and Wang (2010), which affected sporophore formation (Royes, 2002).
Mycelial growth in this study was far slower than the finding of Tan (1981) and Dahmardeh et al. (2010) that the spawn running took three weeks and fruiting bodies appeared after 2–3days. The slower spawn running on cotton seed hull substrate may be due to the high nitrogen, which is known to inhibit mushroom growth in excessive amounts (Table 1). Narain et al. (2008) reported that mushroom mycelia growth and primordial development is dependent on the lignocellulosic materials especially the C:N ratio. Yang (2000) suggested that the C/N ratio favored the primordial formation about 22–30:1, higher C/N ratio favored the mycelial growth, and lower C/N ratio favored the fruiting bodies growth.
Given the physical nature and high porosity of the rice straw or wheat straw, and also the fact that it dries up very fast, it will be advisable to add cotton seed hull when used as mushroom substrate. Similar results have been reported by He et al. (1995) that 20% (w/w) of cotton seed hull was added to rice straw substrate resulting in a 20% increase in mushroom yield, and Wang (2010) reported that 28–56% (w/w) of cotton seed hull was added to wheat substrate resulting in a 15–18% increase in mushroom yield. Yield increases may be due to several factors. Firstly, the increased level of nutrient available at higher rates would provide more energy for mycelial growth and primordial formation. Secondly, supplement of cotton seed hull increased the water-holding capacity, and decreased the mortality of young fruiting bodies due to water shortage.
The variation in stipe length and mushroom cap diameter was observed in the nine substrates used in this study. Relatively smaller mushroom cap diameter and longer mushroom stipe length are undesirable characteristics as for marketable quality. Environmental conditions as well as supplementation of substrates with various additives including nitrogen sources have been reported to improve growth, yield and quality of mushrooms (Royes, 2002; Panjabrao et al., 2007; Onyango et al., 2011). In our study, the supplement of wheat bran and cotton seed hull to rice straw or wheat straw could shorten mushroom stipe length and enlarge mushroom cap diameter, which may result from the supplement changing the physical properties and C/N ratio of rice straw (or wheat straw) substrate. Thus we can conclude that supplementation of cotton seed hull to rice straw (or wheat straw) substrate can increase Pleurotus’s marketable quality.
Using a non-sterilized substrate is an alternative cultivation method to sterilized substrate. In this study, P. ostreatus grown on non-sterilized substrate exhibited faster mycelial growth, shorter total colonization period and time to primordial formation. This may be due to several factors. First, increasing spawn levels was an important factor in simulating mycelial growth, time to primordial formation, and time to first crop of oyster mushroom. The increased level of nutrient available in spawn at higher rates would provide more energy for mycelial growth and development. Second, more inoculum points in non-sterilized substrate would provide faster substrate colonization, thus, more rapid completion of the production cycle (Royse et al., 2004). Finally, the autoclave sterilized substrates were compressed by high pressure for 80min, and air was discharged, thus, the shortage of oxygen may be a reason that slowed the growth of spawn. The relatively high growth and yield on non-sterilized substrate at the expense of undesirable characteristics, i.e. smaller mushroom cap diameter and relatively long mushroom stipe length, when oyster mushroom cultivated on non-sterilized substrate, which also reported by Wang (2010) and Li et al. (2011), is not unexpected. Thus, further research on how to increase the mushroom quality on non-sterilized substrate is necessary.
Footnotes
Peer review under responsibility of King Saud University.
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