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Effect of Substrate Pre-treatment Methods on Oyster Mushroom (Pleurotus

ostreatus) Production

Article  in  International Journal of Agriculture and Biology · January 2012

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INTERNATIONAL JOURNAL OF AGRICULTURE & BIOLOGY
ISSN Print: 1560–8530; ISSN Online: 1814–9596
11–373/MFA/2012/14–2–251–255
http://www.fspublishers.org

Full Length Article  

Effect of Substrate Pre-treatment Methods on Oyster Mushroom
(Pleurotus ostreatus) Production
TAJUDEEN O. OSENI1, SIKHUMBUZO O. DLAMINI, DIANA M. EARNSHAW† AND MICHAEL T. MASARIRAMBI
Horticulture Department, University of Swaziland, P.O. Box M205, Luyengo, Swaziland
†Crop Production Department, University of Swaziland, P.O. Box M205, Luyengo, Swaziland
1
Corresponding author’s e-mail: toseni@ uniswa.sz

ABSTRACT

Among the various cultivated mushrooms, oyster mushroom is easy to cultivate due to its strong enzymatic action towards the
utilization of various kinds of organic substrates. However, pre-treatment of substrates is not an easy job for the cultivation of
mushroom especially in the rural areas. The objective of this study was to devise an easy handy procedure for organic
substrate pre-treatment in the production of oyster mushroom. Substrate pre-treatments by autoclaving at 121°C, hot water
dipping (pasteurisation) in steel drum at 60°C for 2 h and hot water dipping (pasteurisation) in steel drum at 60°C for 3 h were
evaluated. The percent contamination was significantly higher in horse manure compost (70%) compared to sugarcane
bagasse (12.5%). The oyster mushroom took significantly less time to colonise the autoclaved sugarcane bagasse (36 days)
compared to sugar cane bagasse pasteurised for 2 h (64 days). Autoclaved horse compost manure was fully colonised in 42
days, while those pasteurised with hot water at 2 and 3 h failed to colonise due to heavy contamination by Trichoderma
harzianum presumably due to insufficient sterilisation. Despite the shortest days to full colonisation, there was no significant
difference in the yield (410.4 g) and bio-efficiency (82.10%) of autoclaved sugarcane bagasse compared to the yield (301.1 g)
and bio-efficiency (60.22%) of sugarcane bagasse pasteurised in hot water for 3 h. Although, autoclaving was the best method
for substrate pre-treatment, however hot water pasteurisation at 60°C for 3 h of sugarcane bagasse proved to be a viable and
promising method of substrate pre-treatment, which can be adopted to produce a good yield of oyster mushroom especially in
rural areas, where autoclave sterilisation is not feasible. © 2012 Friends Science Publishers

Key Words: Bio-efficiency; Oyster mushroom; Pleurotus ostreatus; Substrate pasteurisation; Yield

INTRODUCTION including paddy straw, maize stalks/cobs, vegetable plant

residues, bagasse etc. (Hassan et al., 2011) and this has been
The Oyster mushroom (Pleurotus ostreatus), is one of reported to influence its growth, yield and composition
the most appreciated mushroom due to its very good taste, (Iqbal et al., 2005; Kimenju et al., 2009; Khare et al., 2010).
high nutritional and medicinal value. It has also been found However, an ideal substrate should contain nitrogen
to exhibit strong anti-inflammatory and immune-modulatory (supplement) and carbohydrates for rapid mushroom growth
properties due to their chemical composition (Lavi et al., (Anonymous, 2008). The cultivation of oyster mushroom is
2010). Mushrooms are a good source of protein, vitamins gaining importance in tropical and subtropical regions due
and minerals (Khan et al., 1981) and are known to have a to its simple way of cultivation and high biological
broad range of uses both as food and medicine (Alice & efficiency (Singh et al., 1990).
Kustudia, 2004). Oyster mushroom is popular and Oyster mushrooms have been traditionally produced
considered a nutritious food in Swaziland but its cultivation using the out - door log technique (Anonymous, 2008),
has long been neglected, because most of the mushrooms thereby eliminating substrate sterilisation. The technology of
consumed locally are picked from the wild. However, large scale mushroom production is a recent innovation and
awareness in oyster mushroom cultivation in Swaziland has the establishment of laboratories for research on mushroom
significantly increased since 2001, when the Queen mother growing and the use of pure spawn culture resulted in rapid
initiated a pilot project on mushroom production in and increased production of mushrooms worldwide (Flegg et
conjunction with the Ministry of Agriculture and al., 1985). The substrates for cultivating edible mushrooms
Cooperatives aimed at poverty alleviation and women e.g. Pleurotus ostreatus, has been reported to require varying
empowerment through job creation in the rural areas degrees of pre-treatment in order to promote growth of the
(Earnshaw, personal communication). mushroom mycelium to the exclusion of other
Oyster mushroom can be grown on various substrates microorganisms (Chang, 2008). The methodology for

To cite this paper: Oseni, T.O., S.O. Dlamini, D.M. Earnshaw and M.T. Masarirambi, 2012. Effect of substrate pre-treatment methods on oyster mushroom
(Pleurotus ostreatus) production. Int. J. Agric. Biol., 14: 251–255
 OSENI et al. / Int. J. Agric. Biol., Vol. 14, No. 2, 2012

substrate preparation described in several studies consists of The cooled bags were inoculated using the prepared
composting agricultural residues, followed by pasteurisation, planting grain spawn of oyster mushroom (Pleurotus
which can be carried out in different ways (Stamets, 1993; ostreatus) under a lamina air flow to minimize
Balasubramanya & Kathe, 1996). contamination at the rate of 5% per bag according to the dry
Sanchez (2010) reported that substrate used for the weight of the substrates. The bags were incubated in dark
oyster mushroom cultivation do not require sterilisation, but chamber for spawn running at a temperature of 28 - 30°C
only pasteurisation, which is less expensive to diminish the until full colonisation by mycelia was attained. The bags
damages produced by different pathogens (bacteria, moulds were later transferred to the mushroom cropping room
or insect pests) on mushroom development and yield. Diana where they were opened to trigger fructification. The
et al. (2006) recommended disinfection of the substratum relative humility inside the growing room was kept as high
before spawning, which should only destroy the competitive as 85-95% by watering the sand floor and spraying the
fungi and not the useful micro organisms. Quimio et al. substrate bags with water twice a day in the morning and
(1990) observed that substrate sterilisation is not ideal since evening.
both beneficial and harmful organisms in the substrate are Mushrooms were harvested as soon as the fruiting
killed, while Miroslawa (1991) recommended maintaining bodies developed and attained their full size above the
the substrate for 24 h at 70°C. Similarly, sterilisation of substrate with sharp knife from each treatment bag.
substrates is not an easy job for the cultivation of mushroom Mushrooms were harvested in three flushes over a 42-day
and the right sterilisation time and temperature depend on period. The parameters recorded were number of
the possible pathogens in a given substrate material (Kwon contaminated bags, days taken for the completion of
& Sik Kim, 2004). substrate colonisation, mushroom pileus diameter (cm),
The mushroom project embarked upon by the stipe length (cm), total mushroom yield (g) and bio
Swaziland World Vision in National Care Points (NCPs) efficiency (BE). The bio efficiency (BE) of mushroom was
around the country in an attempt to provide food for the calculated by using the formula recommended by Chang
orphans and vulnerable children (OVC) was not successful and Miles (1989) as:
due to lack of financial resources to prepare the substrates
% Bio-efficiency (BE) = Fresh weight of mushroom
for mushroom production. Therefore, the objective of this
(g)/Dry weight of substrate (g) X 100.
study was to compare the effectiveness of substrate pre-
treatment methods using autoclave sterilisation and hot The data collected were subjected to analysis of
water pasteurisation on oyster mushroom growth and yield. variance (ANOVA) using MSTAT-C (Nissen, 1989). Mean
separation was done using LSD, where effect showed
MATERIALS AND METHODS significant difference at 5% level of probability.

The study was conducted at the Mushroom laboratory RESULTS

of the Crop Production Department, University of
Swaziland, Luyengo Campus and Malkerns Research The analysis of variance for stipe length, pileus
Station, Malkerns, Swaziland between October, 2010 and diameter, bio-efficiency and mean yield of oyster
January, 2011. Two locally available substrates namely mushroom indicated significant differences among the
sugarcane bagasse and horse manure compost were used in various substrate pre treatment methods (Table I).
this study. To each of the substrate, 20% of wheat bran Number of contaminated bags: There was a significant
supplement was added and thoroughly mixed. The (P<0.05) difference in the number of contaminated bags,
substrates were soaked in clean water for 8-10 h and which ranged from 2 to 40 by green mould (Trichoderma
allowed to imbibe water. The substrates were separately harzianum) and other bacteria during incubation (Table II).
subjected to a short composting procedure using the method The horse manure compost pasteurised either for 2 or 3 h
of Sinden and Hauser (1980). The substrates were divided had the highest number of contaminated bags (40 bags) and
into batches of 500 g each and packed into heat resistant was significantly (P < 0.05) higher than the other treatments
autoclaveable bags of 18 × 14 cm in size, which were and thus abandoned. The autoclaved sugarcane bagasse
fastened at the end using plastic rings. The substrates were exhibited the least contamination (3 bags), which was not
then pre-treated using the following methods viz., steam significantly different from autoclaved horse manure
sterilisation in autoclave at 121°C for 4 h, hot water dipping compost (4 bags) and sugarcane bagasse pasteurised for 3 h
in steel drum at 60°C (pasteurisation) for 2 h and hot water (7 bags).
dipping in steel drum at 60°C (pasteurisation) for 3 h and Number of days taken for full colonisation of substrate:
allowed to cool. The treatments which consisted of two The number of days taken to full colonisation for the
substrates and three pre-treatment methods were arranged in substrate pre-treatments (Table II) differed significantly
a randomised complete block design with four replications (P<0.05). The mycelium failed to colonise the horse
and there were 10 bags per replicate, giving a total of 40 manure compost substrates that were pasteurised for 2 and
bags per treatment. 3 h, respectively presumably due to contamination.

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 EFFECT OF SUBSTRATE PRE-TREATMENT METHODS ON OYSTER MUSHROOM / Int. J. Agric. Biol., Vol. 14, No. 2, 2012

Table I: Analysis of Variance for stipe length, pileus diameter, yield and bio-efficiency of oyster mushroom as
affected by substrate pre treatment
Sources of variation Stipe length Pileus diameter Yield Bio-efficiency
d.f. M.S. F M.S F M.S F M.S F
Replication 3 404.70 2.83 475.40 3.59 1670.13 1.01 417.53 0.94
Treatment 3 1335.71 9.42* 821.80 6.21* 7947.74 4.84* 1986.78 4.47*
Error 9 142.06 132.27 1684.20 444.7
Significant at 5% level

Table II: Effect of substrate pre-treatment on the Yield and bio-efficiency of oyster mushroom: There were
number of contaminated bags and time of colonisation significant (P<0.05) effects of the substrate pre-treatment
of oyster mushroom methods on the average yield of oyster mushroom (Table
IV). The crop of oyster mushroom was harvested in three
Substrate pre-treatment Number of Days to full
contaminated bags colonisation
flushes and the maximum yield was obtained in first flush
Autoclaved Bagasse 3c 36c than the second and third flush, respectively. Maximum
Autoclaved Compost 4c 42c average yield of 410.4 g was obtained from autoclaved
Bagasse pasteurised for 2 h 7b 64a bagasse, while the lowest yield was obtained from bagasse
Compost pasteurised for 2 h 40a 0**
Bagasse pasteurised for 3 h 5bc 52b
pasteurised for 2 h (118.9 g). The yield from bagasse
Compost pasteurised for 3 h 40a 0** pasteurised for 3 h (301.1 g) was higher but not significantly
*Means within the same column followed by the same letter are not different from autoclaved compost manure (209.2 g). The
significantly different at P<0.05 variations observed in yield may, therefore be attributed to
**No colonisation was obtained in these treatments the complexity of substrates in terms of their cellulose
Table III: Effect of substrate pre-treatment on the stipe content resulting from the difference in the rate of
length and pileus diameter of oyster mushroom degradation by the mushroom enzymes as a result of the pre
treatment methods. The Bio-Efficiency (BE) was calculated
Substrate pre-treatment Stipe length (cm) Pileus diameter (cm) to determine how the mushrooms utilized nutrients present
Autoclaved Bagasse 5.48ab* 4.78a
Autoclaved Compost 5.94a 4.84a
in the substrates efficiently. The average bio-efficiency was
Bagasse pasteurised for 2 h 4.78bc 4.70a variable and significantly (p<0.05) different among the
Bagasse pasteurised for 3 h 4.53c 4.37b substrate pre-treatments. Autoclaved sugarcane bagasse had
*Means within the same column followed by the same letter are not the highest BE of 82.10%, which was not significantly
significantly different at P<0.05 different from sugarcane bagasse pasteurised for 3 h
Substrate pre-treatment had variable effects on the duration (60.22%), but significantly higher than autoclaved horse
of full colonisation ranging from 36 to 64 days for manure compost (41.85%). Sugarcane bagasse pasteurised
autoclaved sugarcane bagasse and sugarcane bagasse for 2 h had the lowest BE of 23.78% compared to others.
pasteurised for 3 h, respectively. The days taken to colonise
the sugarcane bagasse sterilised for 2 h were significantly DISCUSSION
longer (P<0.05) than those taken to colonise the sugarcane
bagasse, which was autoclaved and those pasteurised for 3 The ability of a sterilisation method to eliminate
h. However, full colonisation was completed 42 days after substrate contaminants is shown by the presence or absence
inoculation in the autoclaved horse manure compost and of contaminants in the substrate after sterilisation, spawning
was not significantly different from autoclaved sugar cane and incubation. Kurtzman (2010) reported several causes of
bagasse (36 days). mushroom substrate contamination. All the horse manure
Stipe length and pileus diameter growth: The stipe length compost bags pasteurised either for 2 or 3 h were heavily
and pileus diameter development in oyster mushroom were contaminated by green mould and thus abandoned. This
significantly (P<0.05) affected by substrate pre treatment as agreed with Kalberer (1974) who observed that compost
shown in Table III. The mean stipe length ranged from 4.53 was a poor substrate for the growing oyster mushrooms.
cm in sugarcane bagasse pasteurised for 3 h to 5.94 cm in Similarly, insufficient pasteurisation could also be attributed
autoclaved compost manure, but there was no significant to the contamination of the horse manure compost. Kwom
difference in mean stipe length of oyster mushroom in both and Sik Kim (2004) observed that cattle manure contained
autoclaved bagasse and horse compost manure. The pileus high salt levels, which may also increase the risk of
diameter ranged from 4.37 cm in pasteurised bagasse for 3 h contamination. This also concurred with the result obtained
to 4.84 cm in autoclaved horse compost manure. However, by Oei (1996). According to Balasubramanya and Kathe
there was no significant difference (P<0.05) among the (1996), the microorganism species that competed with
pileus diameter in autoclaved horse compost manure, Pleurotus sp. after pasteurisation with hot water (80°C for 2
autoclaved sugarcane bagasse and pasteurised bagasse for 2 h) were the fungi Penicillium sp. and Trichoderma sp.,
h, all of which were significantly larger than in pasteurised probably due to the partial breakdown of cellulose and
bagasse for 3 h. hemicelluloses, thus making them available to competitors.

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 OSENI et al. / Int. J. Agric. Biol., Vol. 14, No. 2, 2012

Table IV: Effect of substrate pre treatment method on the yield of oyster mushroom
SubstratePre-treatment Method Yield of mushroom (g) per 500 g of substrate Average yield (g) in three flushes per 500 g of substrate
1st flush 2nd flush 3rd flush
Autoclaved Bagasse 900.10 325.75 5.33 410.39a*
Autoclaved Compost 256.68 258.40 112.63 209.23bc
Bagasse pasteurised for 2 h 117.83 216.47 22.33 118.88c
Bagasse pasteurised for 3 h 487.00 360.10 56.18 301.08ab
*Means within the same column followed by the same letter are not significantly different at P<0.05
The contamination of the hot water pasteurised substrate that the mushroom received nutrition and energy from the
may have occurred probably due to inadequate temperature abundant free sugars that were present in the bagasse and
and time used during pasteurisation, since the literature is therefore made limited use of the cellulose fraction.
quite variable with reference to these characteristics. Chang Similarly, the average biological efficiency was variable and
and Miles (1989) reported that green mould competes with significantly different among the substrate pre-treatments.
the mushroom for space, nutrients as well as causing This confirms the finding of Mandeel et al. (2005) that B.E
chemical alteration of the substrate, which hinders is highly affected by the quality of the spawn of the
mushroom development. cultivated mushroom strain. Different substrates have been
The substrate pre-treatment had variable effects on used to grow Pleurotus sp. with BE values varying from
days to full colonisation ranging from 36 to 64 days, while 32.10 - 79.18% (Dhanda et al., 1996).
the mycelium failed to colonise the horse manure compost
substrate. Sugar cane bagasse contained high CONCLUSION
polysaccharides, which hastened the fungus growth resulting
in faster colonisation. This concurred with the findings of It can therefore be concluded that hot water dipping of
Iqbal et al. (2005) who reported 37 days for full colonisation sugarcane bagasse in steel drum at 60°C (pasteurisation) for
completion in exotic strains of Pleurotus ostreatus on 3 h despite its low yield and biological efficiency compared
sugarcane bagasse. Result obtained contradicts those of Shah to autoclaved bagasse, would be a viable and promising
et al. (2004); Vetayasuporn et al. (2006); Ponmurugan et al. technique of substrate pre-treatment that can be adopted to
(2007) who obtained full colonisation in Pleurotus ostreatus produce a good yield of oyster mushroom in most rural
in 17 - 20 days on different substrates. Nutritional areas, where autoclave sterilisation may not be feasible.
composition of substrates has been reported to be crucial in
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