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Cultivation of oyster mushroom (Pleurotus ostreatus) on fermented moso bamboo sawdust
⁎Corresponding author. shazwin@utm.my (Shazwin Mat Taib),
-
Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
In this study, the potential of Moso bamboo sawdust as an alternative substrate for the cultivation of oyster mushroom (Pleurotus ostreatus) was investigated. Oyster mushroom was cultivated on 2-months fermented bamboo sawdust (BS) and mixed with rice bran (RB) and sweet potato schochu lees (SPSL) as additional nutrition. The growth condition, morphological properties, nutritional, mineral contents and free amino acid content of mushroom cultivated were evaluated. Based on the results, the total growth days on the bamboo media were between 3 and 7 days shorter than the conventional media. The bamboo media mixed with RB had better yield and fruiting bodies at 97.9 ± 3.9 g/bottle and 33.6 ± 4.2 no/bottle, respectively. Furthermore, the addition of SPSL to BS increased the protein content and decreased the carbohydrate contents of fruit bodies. In addition, the free-amino acids in the fruit bodies from the bamboo media were 1.5 times higher than the conventional media, which potentially added the higher value to usual mushrooms. Hence, oyster mushroom cultivation can be an alternative method to reduce bamboos wastes in Japan and would promote sustainable growth in agricultural industry.
Keywords
Moso bamboo
Oyster mushroom
Rice bran
Sweet potato
Schochu lees
1 Introduction
Moso bamboo (Phyllostachys pubescens) is a large woody bamboo that has ecological, economic and cultural value in Asia and accounts about 70% of the total bamboo growth area (Peng et al., 2013). In western Japan, Moso bamboo forests have been expanding which can raises concerns about possible changes in terrestrial water and carbon cycles (Komatsu et al., 2012). As reported by Tanaka et al. (2013), bamboo shoot skin is used as a preservative container to maintain the quality of tea leaves in China and has been used to wrap rice balls and meats in Japan. Fukuoka prefecture is the major cultivation area of bamboo shoots in Japan. Efficient and sustainable utilization of 5 years old bamboos is a serious concern. Although, the old bamboos are used in traditional craftworks or as pulp, charcoal or livestock feed, but the demand has not exceeded the supply. Bamboo contain cellulose, hemicellulose, and lignin which are similar to conifer sawdust and can be used as media for edible mushroom cultivation. Based on the previous finding, oyster mushroom can be cultivated on variety of substrates that contain lignin and cellulose. Therefore, it have a significant role in managing agricultural wastes which have become a critical issue for disposal (Marlina et al., 2015).
Oyster mushroom is widely studied as it has flavour and contains nutritional and medicinal proprieties. As nutrient source of protein, carbohydrates, vitamins, calcium and iron, it can be used in a variety of applications (Corrêa et al., 2016). For instance, oyster mushroom can be used for medical purposes which can increase the immune power of our body against diseases. Therefore, it can be used as a dietary supplement (Khatun et al., 2015).
Recently, oyster mushroom cultivation on various lignocellulosic materials has been investigated by a number of researchers (Pereira et al., 2017; Postemsky et al., 2017; Sardar et al., 2017). This species is rich in protein sources and minerals such as phosphorus, calcium, iron, potassium and sodium (Szabová et al., 2013). Furthermore, proteins in oyster mushroom has the nutritional requirements of all essential amino acids for adults (Carrasco-González et al., 2017). Oyster mushroom has been cultivated using various agro wastes such as rice straw and wheat straw (Yang et al., 2013; Rezania et al., 2017), date-palm leaves (Alananbeh et al., 2014), empty fruit bunch (Marlina et al., 2015), olive cake (Ananbeh and Almomany, 2005), tomato tuff (Ananbeh and Almomany, 2008), banana leaves and pine needles (Ananbeh, 2003) and sugarcane bagasse (Hasan et al., 2015). Mushroom cultivation on Moso bamboo is an economic approach in agro-industry as the residues is readily available. In mushroom cultivation, typical commercial industry focused on profits gain in terms of most effective, low cost and locally available mushroom substrates materials (Fatriasari et al., 2016). As Moso bamboo can be one of attractive substrates for oyster mushroom cultivation, therefore the aim of this study was to investigate the feasibility of using Moso bamboo as a medium substrate for oyster mushroom cultivation.
2 Materials and methods
2.1 Proximate analysis of medium materials
The components of base materials and nutrient supplements have been evaluated for the following parameters: the moisture content using normal pressure heating and watering method as described by (Alam et al., 2008). Crude protein was determined using Kjeldahl method with a nitrogen/protein conversion factor of 6.25 (Shumaila and Mahpara, 2009). The ash content was measured by method (No. 924.05), and ether extract (EE) using Soxhlet diethyl ether sampling method (No. 920.39), while and crude fiber was calculated by filtration method) (No. 978.10) (AOAC, 1995). Nitrogen-free extract was calculated as described by (Segato et al., 2008).
Minerals composition of phosphorus (P), potassium (K), calcium (Ca) and magnesium (Mg) were determined by atomic absorption spectrometry (AAS) (No. 968.08) (AOAC, 1995). The cedar sawdust was watered and kept for 6 months. Moso bamboo sawdust was fermented for 2 months indoors before media preparation.
2.2 Cultivation test of oyster mushroom
2.2.1 Tested mycelia
Oyster mushroom (Pleurotus ostreatus) mycelia (H67th) which provided by Kinox cooperation, (Sendai Japan) was used for the cultivation. Their outgrowth term was relatively short and they were endowed with antibacterial characteristics (Katya et al., 2016).
2.2.2 Media preparation and inoculation
To determine the effectiveness of bamboo (Phyllostachys pubescencs) sawdust (BS) as an alternative substrate for oyster mushroom cultivation, various combinations of BS, rice bran (RB) Sweet Potato Schochu Lees (SPSL) were used (Table 1). BS was prepared with rice bran/shochu lees as experimental groups, and conifer sawdust (Japanese cedar, Cryptomeria japonica) with rice bran/shochu lees as control groups. The media materials and nutrient supplements were prepared at the rate of 46%: 50% (dry weight) for each group. Then the shell fossil (uncoagulated shell grit aragonite lime) was added at 4% (dry weight) in order to reach to pH 5.5. Media were prepared in a mixer by filling with the base materials and the nutrient supplements and were stirred for 10 min. Then, shell fossil were added to the mixtures and the substrates were humidified with water which resulted in 63% water content in the medium. Water content was calculated after media substrates preparation. Then, the substrates were stirred for 20 min. The prepared media were packed into polypropylene culture pots (capacity: 850 ml, diameter: 58 mm with total weight of 600 g). The packed media were autoclaved at 121 °C for three hours, and cooled at room temperature. Each pot was inoculated with 10 g which is equivalent to 6% (dry weight) oyster mushroom mycelium H67th in a clean room and experiments were conducted with three replication. In this study, CS + RB treatment was considered as a control. CS: Conifer Sawdust, BS: Bamboo Sawdust, SPSL: Sweet Potato Schochu Lees (dry); RB: Rice Bran, SF: Shell Fossil.
Test group
Media composition (dry weight%)
Packed weight (g)
Water content (%)
Base materials
Nutrition
Other
CS
BS
SPSL
RB
SF
1 CS + RB (BL)
46
50
4
600
63.8
2 BS + RB
46
63.0
3 CS + SPSL
50
62.8
4 BS + SPSL
50
63.9
2.2.3 Culturing conditions
The inoculated culture pots were maintained at 22 ± 2 °C temperature and 65 ± 5% humidity in incubation room for 28 days. After the scraping and treatment with tap water (known as Kinkaki treatment) for 3 h, the pots were transferred to a another room and maintained at 14 ± 1 °C and 90 ± 5% humidity to allow for the primordial formation of fruiting bodies. The spawn percentage was maintained at 6% (dry weight). The room was lightened for 8 h/day with 100 lux fluorescent lights.
2.3 Growth conditions
2.3.1 Yield of fruit bodies
The fruiting bodies were harvested when reached to a diameter between 40–50 mm. Various parameters such as the Kinkaki treatment to harvest, total culturing days, yield (raw weights), number of fruit bodies and the biological properties were evaluated.
2.3.2 Analysis of the components of the fruiting bodies
Main components, minerals and heavy metals in the fruiting bodies were evaluated in the same analysis as the media materials mentioned in Section 2.1. Amino acids in fruiting bodies were determined by post-column derivative high performance liquid chromatography (HPLC) (Kim et al., 2009).
2.3.3 Statistical analysis
Completely randomized design was used to arrange the treatments. Three agro-wastes bamboo sawdust (BS), conifer sawdust, sweet potato schochu lees (SPSL) and rice bran (RB), two ratios (46:4%, 50%), 6% spawning rate (of total dry weight in each pot) two replicates were used. The experiment was repeated twice and the data were analysed using Minitab16 Statistical Software.
3 Results and discussion
3.1 Components of raw materials used in mushroom media
Evaluation of component properties is essential to observe the phenomenon of media composition before and after cultivation. Table 2 shows the component properties of raw materials derived from bamboo sawdust (BS), conifer sawdust, sweet potato schochu lees (SPSL) and rice bran (RB). As expected, BS as the base material had more nitrogen-free extract (NFE) (45.1 g/100 g) and less crude fiber (50.8 g/100 g) when compared to conifer sawdust. BS had high amount of NFE which consists plenty of single oligosaccharides and starch that are easily decomposed by mycelia (Zullaikah et al., 2015). The crude protein content of conifer sawdust grew only 1.1 g/100 g compared to BS which is 1.5 g/100 g. Compared to bamboo sawdust, component properties of conifer sawdust were quite low amount except for ether extract (1.1 g/100 g), crude fiber (75.9 g/100 g), crude ash (2.5 g/100 g), and calcium (Ca) (265 g/100 g). In addition, BS comprised significant amount of potassium (K) 50.8 mg/100 g and lower amount of Ca (24 g/100 g).
Materials
CP
EE
CF
CA
NFE
P
K
Ca
Mg
(g/100 g dry weight)
(mg/100 g dry weight)
Bamboo Sawdust
1.5
0.9
50.8
1.7
45.1
24
508
24
39
Conifer Sawdust
1.1
1.1
75.9
2.5
19.4
8.6
5.3
265
32
Sweet Potato Shochu Lees (dry)
24.7
3.9
11.8
4.7
54.9
364
1390
717
98
Rice Bran
15.8
21.8
8.3
10.5
43.6
860
1697
40
950
In order to obtain optimized composition for oyster mushroom cultivation, nutrient supplements is needed to stabilize the macro and micronutrients of base materials. In terms of nutrient supplements, SPSL had high level of crude protein (24.7 g/100 g), crude fiber (11.8 g/100 g), NFE (54.9 g/100 g), and Ca (717 g/100 g). Shochu is a traditional Japanese distilled liquor, made by rice koji (Aspergillus kawachii), which is koji mould grown on rice grain. Rice koji is an essential ingredient of Japanese liquors such as shochu and plays as a source of enzyme to degrade starch (Shiraishi et al., 2016). In contrast, RB contains higher amount of ether extract (21.8 g/100 g), crude ash (10.5 g/100 g), phosphorus (P) (860 g/100 g), K (1,697 g/100 g) and magnesium (Mg) (950 g/100 g) compared to SPSL. Although, both materials contain enough amount of K as to allow for the formation of fruit bodies, the quantity of other mineral elements are much different.
3.2 Cultivation of oyster mushrooms
Table 3 demonstrates the growth conditions for different test groups. The growth days for mycelial were more in the shochu lees/bamboo sawdust groups (Group 4) compared to the control group. This is due to the presence of some mycelial growth inhibitors such as tannin and phenol in bamboos (Nirmala et al., 2014), and fatty acid ester in shochu lees (Shiraishi et al., 2016). However, after the incubation, both the days from Kinkaki treatment to harvest and the total growth days were shortened to 3–7 days compared to the control group. Mycelia growth successfully accelerated with the presence of the nucleic acid-related substances (Ohga et al., 2003). SPSL contain 1.4 times the nucleic acid-related substances of RB since it contains koji from shochu lees and yeast. BS which have been fermented for 2 months, contains microbe-derived nucleic acid substances, which seem to have shortened the growth. The mycelial growth inhibitors in Group 4 must have been decomposed by mycelia during the incubation. Values are mean ± standard deviation. **Abbreviations (CS: Conifer Sawdust, BS: Bamboo Sawdust, SPSL: Sweet Potato Schochu Lees (dry), RB: Rice Bran Significant difference between the values with different alphabets at the 5% level (Tukey's test) (Tukey, 1949).
Test group
Days for mycelial growth
Incubation days
Days from Kinkaki treatment to harvest
Total growth days
1 CS + RB (BL)
15.4 ± 0.4a
28
20.2 ± 2.4c
48.2 ± 2.4c
2 BS + RB
18.0 ± 0.7b
16.8 ± 1.6bc
44.8 ± 1.6bc
3 CS + SPSL
17.6 ± 0.5b
12.0 ± 0.7a
40.0 ± 0.7a
4 BS + SPSL
18.2 ± 0.4b
13.2 ± 0.8ab
41.2 ± 0.8ab
Table 4 shows the morphological properties, the number, and the yield (raw) of mushrooms from each group. In test group 2, the samples showed high yield of mushroom compared to other mixtures where the yield was 97.9 ± 3.9 g and number of fruiting bodies were 33.6 ± 4.2. Morphological properties did not show any remarkable differences from the control group except the top layers were slightly thinner (9.1 ± 1.2 mm). SPSL, one of Japanese local food wastes was as a qualified nutrient supplement for growing edible mushrooms with a high yield (Yamauchi et al., 2011, 2013). Present of SPSL in the media had significant yield for mushrooms which test group 3 and 4 obtained higher yield compared to control group (test group 1) at 91.5 ± 7.4 g and 94.2 ± 3.9 g, respectively. Significant difference between the values with different alphabets at the 5% level (Tukey's test). Values are mean ± standard deviation.
Test groups
Quality
Maximum diameter of Pileus
Maximum thickness of Pileus
Maximum diameter of Stalk
Fruiting bodies
Yield (raw)
(mm)
(mm)
(mm)
(No./bottle)
(g/bottle)
1 CS + RB (BL)
45.5 ± 1.4
11.7 ± 1.2ab
14.2 ± 1.0b
30.8 ± 7.0
90.3 ± 6.8
2 BS + RB
44.1 ± 0.8
9.1 ± 1.2a
10.4 ± 2.5a
33.6 ± 4.2
97.9 ± 3.9
3 CS + SPSL
48.4 ± 4.1
13.6 ± 2.1bc
10.8 ± 0.6ab
29.4 ± 4.0
91.5 ± 7.4
4 BS + SPSL
49.1 ± 4.4
16.0 ± 1.6c
12.1 ± 0.9ab
27.0 ± 2.4
94.2 ± 3.9
Table 5 shows the analysis of the main components and the minerals of fruit bodies from each test group. In test groups 3 and 4 with SPSL as a nutrient supplement, the protein content in the fruit bodies was higher than RB as a nutrient supplement by 43.8 g and 31.1 g, respectively. Meanwhile, the carbohydrate content slightly lower than media supplemented by RB (Group 1 and 2). Protein content in the oyster mushroom’s fruit bodies increased when they were grown with high-protein nutrient supplements compared to control groups with relatively less protein nutrients (Kawai et al., 1994). Protein content in SPSL was 24.7 g/100 g (dry) while in RB was 15.8 g/100 g (dry). Lower amount of carbohydrate content might be caused by the absorption of nitrogen compounds in SPSL. Seventh Revised Edition*
Test group
Protein
Lipids
Carbohydrate
Ash
P
K
Ca
Mg
(g/100 g dry weight)
(mg/100 g dry weight)
1 CS + RB (BL)
35.1
1.8
56.8
6.3
1171
2523
N.D
117
2 BS + RB
33.6
1.8
57.5
7.1
1150
2655
N.D
106
3 CS + SPSL
41.1
3.2
48.3
7.4
1474
2842
N.D
168
4 BS + SPSL
43.8
3.4
43.8
9.0
1573
3707
N.D
146
31.1
2.8
58.6
7.5
943
3208
9.4
142
The minerals in the fruit bodies for all test groups were K > P > Mg in decreasing order of abundance. The value of K was 38–41% of the ash. One of characteristics of mushrooms is possess high amount of potassium and have been distributed unevenly within cap, stipe, spore-forming part and spore (Kalac, 2012). Ca was not found in all test groups of the fruit bodies. The use of BS as a replacement for the conifer sawdust did not affect the main components nor the minerals of fruit bodies.
Amino acids are generally known as protein regulation metabolism which effectively repairs human body metabolisms (Murakami et al., 2012). All test groups were analyzed for free-amino acids contents in the fruit bodies of oyster mushrooms (Table 6). The amount of free-amino acids in the samples from test groups 2 and 4 was 1.5 times higher than the control group (Group 1). The details on the free-amino acids were as follows: Alanine (Ala) and Glutamic acid (Glu), Arginine (Arg) and Glutamine (Gln) were higher than other amino acids, since they are generated by transamination at an early stage. In the test groups 2 and 4, Isoleucine (Ile), Valine (Val), Threonine (Thr), Phenylalanine (Phe) appeared to be the essential amino acids; Serine (Ser), Glutamic acid (Glu), Proline (Pro), Tyrosine (Tyr), Glutamine (Gln), Aspartic acid (Asp) are non-essential amino acids and are higher than Group 1. Optimizing the amino acid supply required for body protein growth with limited impacts on the environment is a generic imperative in all production systems (Kaushik and Seiliez, 2010). Based on the results it is concluded that BS promotes the production of free amino acids in oyster mushrooms compared to conifer sawdust substrates. *All the experiments were conducted in duplicate.
Test groups
Essential
Semi essential
Non-essential
Total
Leu
Met
Ile
Val
Thr
Phe
Lys
His
Arg
Gly
Ser
Glu
Pro
Tyr
Gln
Ala
Asp
(mg/100 g dry weight)
1CS + RB(BL)
316
84
155
201
234
272
398
138
458
219
269
387
216
248
351
1051
39
5035
2 BS + RB
378
169
349
481
435
463
401
211
442
219
504
513
565
435
754
1036
145
7500
3 CS + SPSL
252
181
135
180
189
216
171
225
1036
171
243
955
126
279
1048
946
54
6407
4 BS + SPSL
453
98
301
355
398
402
491
218
599
370
406
894
301
407
602
1126
111
7532
4 Conclusion
Conifer sawdust or broad-leaved tree sawdust is generally used for mushroom cultivations. Bamboo sawdust contains more nitrogen-free extracts (NFE) and less crude fiber compared to conifer sawdust. Based on the obtained results, bamboo sawdust with sweet potato shochu lees or mixture of rice bran shortened the total growth days by 3–7 days compared to the control group. Bamboo sawdust as the substitution for conifer sawdust did not affect the main components or the mineral compositions in the fruit bodies. The oyster mushrooms grown in the media with bamboo sawdust contained higher amount of free-amino acids than those from the control group of conifer sawdust. From these results, we concluded that bamboo sawdust can be used as the base material for the oyster mushroom cultivation. In future, the investigation of Moso bamboo sawdust could be applied in Malaysia, Thailand and Indonesia with their subtropical climate in order to extend their usage.
Acknowledgment
The authors acknowledged the grants FRGS - solvent extraction and precipitation of nanosized titanium dioxide and aluminum oxide from water treatment sludge (R.J130000.7809.4F472) and GUP - an exploratory study of river water treatment using different adsorbents for the removal of recalcitrant organic compounds, nutrients and oil and grease (Q.J130000.2517.10H25).
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