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J. Microbiol. Biotech. Res., 2011, 1 (3):1-14

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ISSN : 2231 3168 CODEN (USA) : JMBRB4

Cultivation of Pleurotus ostreatus: An edible mushroom from agro base waste products
1*

Amuneke E. H., 2*Dike K. S., and 3Ogbulie J. N.,

Laboratory Services, Ministry of Petroleum and Environment, Owerri, Imo State. Nigeria Department of Applied Microbiology and Brewing, Nnamdi Azikiwe University, Awka, Nigeria. 3 Department of Industrial Microbiology, Federal university of Technology,Owerri,Imo state, Nigeria
2

______________________________________________________________________________ ABSTRACT Pleurotus ostreatus an edible mushroom was cultivated from agricultural wastes such as saw dust, cassava peeel, cotton waste, dry plaintain, palm oil chaff, and vegetable. Standard microbiological and chemical methods were adopted to determine the microbial and proximate quality of the substrates. The cultivation of this species involved isolation of the mushroom, preparation of the spawn and substrate, spawning, incubating, cropping, harvesting and packaging. The study showed that sterilization and watering of the mushroom bag was a limiting factor in the growth of the mushroom. Organisms such as Bacillus spp,Mucor spp and Rhizopus spp were implicated during substrate preparation and fruiting of the mushroom. All the substrates supported the growth of fungi except vegetable. The results of the proximate analysis indicated that palm oil cheaf had the highest level of the moisture content, ethyl ether, carbohydrate content and ash content, while saw dust, dry plaintain leaf, cotton waste, cassava peel had an appreciable level of moisture content, ethyl ether,ash content and carbohydrate. Key words: Pleurotus ostreatus, mushroom, cultivation, proximate ______________________________________________________________________________ INTRODUCTION Man has been a mushroom fancier for a very long time. His attention must have been attracted to them by the unusual shape of the fruit bodies, which suddenly appear after rain in striking quantities in fields and wood lands. These fruit bodies which are known today as mushrooms appear white and have a short life span. Some species are edible while some others are non edible [2,3],

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Dike K. S et al J. Microbiol. Biotech. Res., 2011, 1 (3):1-14 ______________________________________________________________________________ Mushrooms belong to the class of Basidiomycetes and order Agaricales. They do not posses chlorophyll like green plants for manufacturing their food but for their growth and development they require preformed food like smaller broken down molecules of cellulose and starch [4] in its simplest form, the life cycle of a mushroom may be traced from - a spore which under favourable conditions germinates to form a mass of branched hyphae of mycelia with colonies in a substrate. This represents the vegetative stage of its growth. When a given substrate is fully colonized, the vegetative growth ceases. Typically some of hyphae form primordial or fundament which is the beginnings of the productive stage [3] This develops further to form the stipes (stalk), the pileus (cap) of the fruit body, which when mature exposes the gill, tissue or generative tissue on the under side, from which spores are liberated, so that the life cycle is perpetuated. Many fungi that form mushroom exist in mycorrhizal with trees, and this is one of the reasons why the forest is often the target for mushroom hunters. Many have learnt through the ages, by trials and error, to identify the edible and inedible mushrooms. In many cases some inedible ones resemble the edible types and are eaten without adverse effect (10) However, there have been occasional accidents of consuming poisonous species leading to death or serious illness. Some kinds of mushrooms such as Amanita muscana had been reported to produce intense excitement and hallucination to the consumer ([14, 12, 21] Mushrooms have now been recognized universally as food and are grown on commercial scale in many part of the world including Nigeria.(10)observed that this fungus is common in Nigeria and often found growing around the African breadfruit (Treculia africana). In Nigerian, the most prized edible species are Pleurofus, Termifomyces, Tricholoma and Volvariella .However, mushrooms hold a unique place in the world today. Human population expands by 2.1% representing a rise of about 75 million people per year. Thus food production has to keep pace with population increase. Mushroom along with yeast are referred to as alternative source of food. [6, 2, 11] According to (11)edible mushroom (dry) contain about 19-40% protein; that is its protein content is twice that of vegetable and four times that of oranges, and they are rich with vitamins, and minerals, less percent of unsaturated fatty acid and carbohydrate which makes it so ideal for diabetic and the obesity patient. Most mushroom has exceptional medicinal potentials and properties; curative and prophylactic especially in diseases such as high blood pressure, asthma, respiratory tracts infection, anaemia, hepatitis, cancer, tumour, e.t.c. [15,17,16] Mushrooms cultivation also serves as the most efficient and economically -viable biotechnology for the conversion of long-cellulose waste materials into high-quality protein food and this will naturally open up new job opportunities especially in rural areas and may be prepackage by food industry and exported to other countries as food conditions and for revenue generation. Pleurotus is the scientific name for Oyster mushroom. In many parts of India; it is known as Dhin [9, 10, 15].It belongs to the family Tricholomataceae which includes many species such as P. flobellotus P. sojar - caju, P. eryngii, P. osfreafies, P. floride and P. sapidus. (9) described P. eurofus species as being convex, becoming plane or occasionally funnel shaped, cap 4-15cm in 2 Available online at www.scholarsresearchlibrary.com

Dike K. S et al J. Microbiol. Biotech. Res., 2011, 1 (3):1-14 ______________________________________________________________________________ diameter or kidney shaped (If growing on the tops of logs), the gills running down the stem (if a stem is present), whitish or with a gray tinge, usually absent or rudimentary, when the mushroom is growing from the side of a log or tress. When it grows on the tops of logs or branches, or at an angle however, it may develop a substantial and thick stem that is dry and slightly hairy near the base. The white flesh has a pleasant taste, but its form texture that makes it popular for eating. [12] divided the genus into four sections with thirty-nine species. However, the species especially in the section Pleurotus is controversial and may be considered tentative, for instance, some North America author call the Oyster mushroom that gives a lilac to grayish - tinge spore print on white paper P. sapiduskalchbr form but otherwise not distinguishable from P. sapidus to be P. ostreatus.Pleurotus species appear during the period from March to early November in tropical rain forest and its require a temperature of 20-30c (23+3c being optimum) both for its vegetative growth and reproductive phase [4,5] According to[14] some specific Microorganisms are important in the preparation of the compost and initiation of fruiting in mushroom. This present work is aimed at assessing the microbiological and proximate analysis of mushroom production from Agro-based waste, a study which has great economic importance for income generation and for improving protein content of food. MATERIALS AND METHODS Source of substrates and materials The substrates (saw dust, cassava peels, cotton waste, dry plantain leave, palm oil chaff and vegetable.) were bought from a hawker at Ekeukwu Owerri market, the substrates were covered with white transparent polythene bag and transported to the National roots crops Research Institute Laboratory Umudike Umuahia for storage in the refrigerator at 4C prior to microbiological and proximate analysis. The facilities of the Central Service Laboratory of the National Roots Crops Research institute Umudike, Abia State were used for both the microbiological and proximate analysis of the sample. Preparation of substrates The eighteen substrates; Group 1 (six substrates before sterilization), Group 2 (Six substrates after sterilization) and Group 3 (Six substrates after harvest) were examined and any extraneous material (i.e. dirt) was removed. Then Group 1, Group 2 and Group 3 were used for microbiological assay and only Group 2 and Group 3 was used for proximate analysis. Substrates for proximate analysis A part of substrates in Group 2 and 3 was spread in the oven at 65c until it was dried to constant weight. Sample was grind in an Author Thomas laboratory mill and sieved through a 1mm sieve to obtain a powdered processed sample, which was used for determination of the proximate composition. Substrates for microbiological analysis A part of substrates in Group 1, 2 and 3 were used directly for microbiological analysis. It was used fresh to avoid loss of some members of the microbial flora during drying and also to avoid contaminant. Twenty - grammes (20g) of the sample was measured and blended in a sterile blender (sterilized by UV- irradiation) using l00mls of sterile distilled water. After blending, it

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Dike K. S et al J. Microbiol. Biotech. Res., 2011, 1 (3):1-14 ______________________________________________________________________________ was transformed to a sterile conical flask and cooled. An aliquot of each prepared substrates (l.0) was used for microbial analysis. Microbiological analysis of sample Determination of microbiological load (bacteria and fungi load) The method of the International Commission on Microbiological Specification for Foods, ICMSF (1978) was adopted and used. One milliliter (1m) of the prepared Group 1,2 and 3 substrates was diluted in 9ml of sterile distilled water (diluents) and mixed vigorously by shaking. 1ml o f the resultant mixture was aseptically transformed to 9mls of sterile water in a test tube. This action was carried out under sterile aseptic conditions. The dilution was continued serially until the 4th dilution was attained (i.e. 10-4)An aliquot of (0.lm) of the 4th dilution was inoculated into a sterile Potato Dextrose Agar (PDA) and Nutrient Agar (NA) plates respectively. The spread plate technique as illustrated by (15) was employed. A flamed glass hockeystick shaped rod was used to spread the inoculum evenly over the surface of the agar in the plate. The arrangement was done for each of the group 1, 2 and 3 substrates. The PDA culture plate was incubated at room temperature for 2 to 5 days, while the NA culture plates were incubated at 37c for 24-48hrs.All the plates were observed daily.The number of colonies formed in each culture plate was counted using the Gallenkamp electronic colony counter. A mean of the counts from the each plate was obtained and multiplied with the appropriate dilution factor to obtain the microbial loads as the total viable microbial colonies per unit weight of the sample expressed as the colony forming unit (CPU) per gramme of the sample. It was calculated using the formula: Cfu/g 1/w x NXD Where W = Weight of sample analyzed N = Average Colonies per plate. D = Dilution factor In all cases, the microbial counts were taken from plate supporting not more than 300 colonies (Pelczar and Chain, 1977) Identification of bacteria Isolates were identified according to [10] and [20] by cell and colony morphology, Gram staining, catalase test, growth at 15OC and 45OC, spore staining, motility test and other biochemical tests like oxidase, indole production, methyl red, Voges-proskauer, liberation of ammonia from arginine, growth in 4% broth, hydrogen sulphide production, growth at 4% NaCl, casein hydrolysis and carbohydrate fermentation Identification of fungal isolates Fungi isolates were identified primarily based on their colonial features and microscopic examination. The slide culture technique adopted by [15] was employed. The isolates were cultured in a semi-solid agar medium and grown on a microscopic slide between two ridges of 4 Available online at www.scholarsresearchlibrary.com

Dike K. S et al J. Microbiol. Biotech. Res., 2011, 1 (3):1-14 ______________________________________________________________________________ wax covered with a cover slip. After incubation, the culture was examined directly under the microscope and its cultural features observed and recorded. Portions of the culture were also made on a slide in lactophenol cotton blue, and on examination detailed features of each fungus isolate was observed and recorded. Findings were crosschecked with features present in [5] Mushroom Cultivation Preparation of Grain Spawn The millet grains were thorough washed and soaked for 24hours in water, and then sieved. After this, the grains were filled halfway (to create air space for welling and shaking) into heat resistant bottle at 121c for 2hrs, for two consecutive days using the autoclave. On cooling, the bottled grains were inoculated with 6cm mycelial discs and incubated at 27+lc for about 2 weeks. Within this incubation period, the bottles were shaken at three-day intervals to avoid tissue clumping and also to aid even and rapid colonization of the grains by the mycelium. Preparation of Substrate for Spawning The six different substrates were spread on different trays for about One week and a regular turning interval of 3days was observed to ensure equal temperature condition of the compost. After these stages, the substrate were differently mixed with lime and soaked in water, excess water was squeezed out of it, and then filled in heat resistant bag. A plastic cooler was then filled onto each bag and stoppered with a cotton plug. The substrates were sterilized for 3hours, at a temperature of 80c in a dried fire-heated drum. The sterilized substrates were allowed to cool overnight in preparation for inoculation. Spawning, incubation and cropping On cooling, 150g of the grain spawn were inoculated into each bag and mixing thoroughly with the substrate. The inoculated bags were transferred to a clean and disinfected incubation room for 2 to 3 weeks. A dark environment and room temperature of 24-28c was maintain during incubation to enhance the quick colonization of the substrate. After full colonization of the bags, they were transferred to the cropping room, whose environment was kept illuminated by sunlight through the improvised windows and a temperature and humidity of 28c and 75-85% were maintained through adequate watering of the mushroom. Harvesting Formation of a complete mushroom occurred one week after the colonized bag has been transferred into the cropping room. The appearance of those mushrooms occurs in flush and about 3 flushes occur in a week continuously for a period of four weeks. A clean scalpel was used to detach the fruitbodies at the base of the stipe from the bags. Weight and numbers of the fruitbodies were recorded per replicate bag before the fruitbodies were consumed. Determination of proximate composition Determination of moisture content The moisture content was determined by the gravimetric method of [1] One gramme of each substrate Group 2 and 3 was measured separately into previously weighed moisture can. It was 5 Available online at www.scholarsresearchlibrary.com

Dike K. S et al J. Microbiol. Biotech. Res., 2011, 1 (3):1-14 ______________________________________________________________________________ then dried in the oven at 105c for 3hrs, cooled in a dessicator and re-weighed. The cooled sample was returned to the oven for further drying. Drying, cooling and weighing were repeated at Ihr intervals until no further reduction in the weights was obtained (constant weight) The weight of moisture loss was determined and expressed as a percentage of the sample analyzed. It was calculated using this formula; %Moisture content = W2-W3 x 100 _______ W2-W1

Where W1 = W2 = W3 =

weight of empty can weight empty can + sample before drying. weight of empty can + sample after drying.

Determination of protein content The protein content of the sample was determined by the Kjeldahl method James (1995) and Chang (2003) in which the nitrogen content was determined and multiplied with 6.25 to obtain the protein content. About 0.2 g of the each processed group 2&3 separately was mixed with l0 mls of H2S04, and a table of selenium catalyst was added. The mixture was heated under a fume cupboard (digestion) until a clear solution was obtained. The digest was diluted to 100ml and used for analysis. 10mls the digest was contained equal amount of 45% NaOH solution in a Kjeldahl apparatus. The mixture was distilled and collected into 10ml of 4% boric acid solution in which 3 drops of mixture indicator (Methy red indicator and bromocressol green) was added. A total of 50ml of distillate was collected and filtrated against 0.02N H2S04 solution. A reagent blank was also digested, distilled and filtrated. The N2 content was calculated using the formular below: % N2 [W W N V1 Va T Klk % Protein = 100 Va] = = = = = = Weight of sample Normality of the titrant Total digest volume Volume of digest distilled Litre of sample Reagent Blank = 100x[NX 14 X V1 T-Blk.]

% Nitrogen x 6.25

Determination of ash content This was done by the furnace incineration gravimetric method [9] 2.0g of each processed group 2 and 3 substrates was separately added into a weighed porcelain crucible. It was then count to

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Dike K. S et al J. Microbiol. Biotech. Res., 2011, 1 (3):1-14 ______________________________________________________________________________ ashes in a muffle furnace at 55c. After cooling in a dessicator, it was re-weighed and the weight of the ash was obtained and expressed as a percentage of the weight of sample analyzed. Calculation of percentage ash was based on the formular below:% Ash = 100 (W2 W1) _____________ W Weight of empty crucible Weight of crucible + ashes Weight of sample

W2 W2 W

= = =

Determination of fat content The soxhlet solvent extraction method of James [9] was employed for determination of the fat content. 2g of each processed Group 2 and 3 substrate was separately wrapped in a porous filter paper and put in a thimble. The thimble was then placed in a soxhlet reflux flask and mounted into a weighed extraction flask containing 200mls of petroleum ether. The upper end of the reflux flask was converted to a condenser. When heated the solvent condenses in to the reflux flask and cover the sample until the flask is filed up and siphoned over carrying oil (fat) extract down to the boiling flask. The process was allowed to go on repeatedly for about 4hours before the defatted sample was removed and kept for crude analysis. The solvent was recovered and the flask with its oil extract was dried in the oven at 60c for 30 minutes, cooled in a dessicator and re-weighed to obtain the weight of the oil extract (fat), which was then expressed as a percentage of the sample analyzed. The percentage fat content was calculated using the formular below :% Fat = 100 (W2 W1) x 100 Weight of sample W1 W2 = = 1

Weight of extraction flask Weight of flask + oil (fat) extract.

Determination of crude fiber This was determined by the Weende gravimetric method.2.0g of each processed Group 2 and 3 substrates was separately defatted and then boiled in 200ml of 1.25% H2S04 for 30 minutes under reflux. The mixture was then washed in hot water using a two-fold muslin cloth to retain the particles. The sample was boiled again in 1.25% NaOH solution for another 30 minutes, washed as before and allowed to dry in the oven at 105c for 4hours. This sample was cooled in a dessicator and weight. After weighting, it was burnt to ashes in a furnace, cooled and weighed. The fibre content in percentage was calculated using the formulae below:% W2 Crude fibre = W2 W3 x 100 W Weight of empty crucible + sample after boiling, washing and drying. 7 Available online at www.scholarsresearchlibrary.com =

Dike K. S et al J. Microbiol. Biotech. Res., 2011, 1 (3):1-14 ______________________________________________________________________________ W3 W = = Weight of crucible + sample after ashing Weight of sample

Determination of pH In determining the pH of the substrates directly, pH measurement was carried out using a pH meter. 1g was measured out from each of the substrate before inoculation and soaked in 10ml of distilled water contained in a clean glass beaker. The substrates were allowed to stand for 10 minutes while stirring after 2minutes intervals. The pH meter (Hannan electronic pH meter) was allowed to equilibrate for about 5mins after its set up. It was standardized with buffering solutions of pH 4 and 7 respectively. For the substrate pH readings, the meter electrode was inserted into the each substrate, allowed to stabilize and the pH values recorded directly. RESULTS Table 1 and 2 shows the proximate quality of substrate before inoculation and harvested substrate of sawdust, cassava peel, cotton waste, palm oil chaff, dry plantain leaf and vegetable leaf. Palm oil chaff had the highest level of moisture content and least level of the moisture (dm) content, CHO content, Ash (ASH) content, Crude fibre (CF) content and EE content. The vegetable leaf had the least level of moisture content and the highest level of dm content, EE content, ASH content and CHO content. While the sawdust, dry plantain leaf, cotton waste, cassava peel had an appreciable level of moisture content, DM content, EE content, ASH content and CHO content. Statistical analysis shows that the cultivation of mushroom on the substrate, had a significantly effect on the Ash content and crude protein. Microbial analysis as shown in table 3, 4, 5, 6, 7 and 8 reveals high level of hetertrophic bacterial count in the unsterlized substrate (cassava peel 9.6 x 106 cells/m/s) and harvested substrate (cassava peel 7.2x106 cells/m/s) while lower bacterial count was found in sterilized substrate ( cassava peel 5.7xl06 cells/m/s) and the highest level of hetertrophic fungi count predominated the unsterilized substrate ( cassava peel 3.2xl04 cells/m/s) and harvested substrate cassava peel (2.6xl04 cells/m/s) while lower fungi count was found in the sterilized substrate (cassava peel 1.8xl03 cells/m/s). Fungi belonging to the genera Mucor spp, Rhizopus spp, Fusarium spp, Asperigillus spp, Penicillium spp and bacteria such as Bacillius spp, Klebsiella spp, Micrococcus spp, Staphylococcus spp and Proteus spp were isolated respectively from unsterilised, sterilized and harvested substrate. The isolates Mucor spp, Rhizopus spp, Proteus spp and Staphylococcus spp were mostly found in unsterilised and harvested substrate while the isolates of Mucor spp and Micrococcus spp are found mostly on sterilized substrate. In Table 10 the initial pH of all the substrates before inoculation of the fungi was alkaline. After colonization, degradation and utilization of the substrate materials by the fungi mycelium, the PH of the substrates dropped below the neutral point to the acid values of 3.6 to 4.2.

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Dike K. S et al J. Microbiol. Biotech. Res., 2011, 1 (3):1-14 ______________________________________________________________________________

Table 1 Proximate composition of the unsterilised substrates used in the cultivation of Pleurotus ostreatus Waste Cotton Waste Cassava Peel Palm Oil Chaff Vegetable Sawdust Dry Plantain Leaf MC 50.26 51.02 54.34 59.30 74.64 74.60 48.74 48.68 61.74 60.80 47.78 49.24 DM 49.74 48.98 45.66 45.70 25.36 25.40 51.26 51.32 38.26 39.20 52.72 50.76 PC 4.56 4.56 4.84 4.86 2.43 2.45 7.60 7.63 4.50 4.53 6.84 6.84 EE 3.84 3.86 3.50 3.50 4.87 4.84 2.36 2.34 3.20 3.18 3.06 3.07 CF 21.74 21.76 18.74 18.71 9.52 9.52 14.78 14.74 8.25 8.16 12.28 12.28 ASH 12.16 12.20 7.20 7.18 4.80 4.82 13.28 13.36 6.69 6.75 11.24 11.30 CHO 57.70 57.62 65.72 65.75 78.38 78.37 61.98 61.93 77.37 77.38 66.58 66.51

MC: Moisture content; DM: Dry mass; PC: Protein content; EE: Ethyl ether; CF: Crude fibre; ASH: Ash content CHO: Carbohydrate content Table 2.Proximate composition of the sterilized substrates used in the cultivation of Pleurotus ostreatus after harvest Waste Cotton Waste Cassava Peel Palm Oil Chaff Vegetable Sawdust Dry Plantain Leaf MC 49.76 49.76 54.28 54.32 69.76 69.76 49.45 49.18 63.78 63.74 48.74 48.60 DM 50.24 50.24 49.72 45.68 30.24 30.24 50.55 50.82 36.22 36.26 51.62 51.40 PC 4.24 4.18 4.38 4.38 2.30 2.32 7.60 6.24 4.18 4.18 5.80 5.76 EE 3.80 3.82 1.46 1.50 3.96 3.96 2.36 2.16 1.25 1.28 2.84 2.92 CF 20.64 19.96 15.94 15.92 9.78 9.76 14.78 15.22 8.74 8.74 13.74 13.68 ASH CHO 11.94 59.39 11.86 60.18 15.70 63.52 15.68 65.56 5.12 78.84 5.16 78.80 13.28 61.98 2.34 69.04 4.68 81.15 4.68 81.12 9.28 68.34 9.34 68.30

Table 3.Total fungal viable count of the substrates after harvest S/NO Cotton waste Cassava peel Palm oil cheaf Vegetable Plantain leaf Sawdust ANC ORGANISM IDENTIFIED 1.3xl04 l.8xl04 1.2xl04 1.4xl04 1.6xl04 1.7xl04 A,B,E A,B A,C,D E, B B,C,E A, D

A:Mucor spp; B:Rhizopus spp; C:Fusarium spp; D:Penicillium spp

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Dike K. S et al J. Microbiol. Biotech. Res., 2011, 1 (3):1-14 ______________________________________________________________________________

Table 4. Fungal load of the unsterilised substrates S/NO Cotton waste Cassava peel Palm oil cheaf Vegetable Plantain leaf Sawdust ANC 2.5xl04 3.28xl04 l.SxlO4 5.6xl0 2.9xl0
4 4

ORGANISM IDENTIFIED AB, B,A A,D C, B C,B A, D,B

3.1xl04

Table 5. The fungal load of the sterilized substrates S/NO Cotton waste Cassava peel Palm oil cheaf Vegetable Plantain leaf Sawdust ANC 02.1xl04 0.18xl04 0.08xl04 0.16xl04 0.17xl04 0.8xl0
4

ORGANISM IDENTIFIED AB, B,A A,D C, B C,B A

Table 6.Fungal load of the sterilized substrates S/NO ANC ORGANISM IDENTIFIED H,J,I F,H G,F I,F,H H,F,I I,H,G

Cotton waste 4.2xl08 Cassava peel 5.6xl08 Palm oil chaff 4.8xl0 Vegetable
8

7.6xl08
4

Plantain leaf 4.8xl0 Sawdust

5.1xl08

F:Bacillus spp G:Micrococcus spp H:Staphylococcus spp I:Proteus spp

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Dike K. S et al J. Microbiol. Biotech. Res., 2011, 1 (3):1-14 ______________________________________________________________________________

Table 7.Fungal load of the sterilized substrates S/NO ANC ORGANISM IDENTIFIED Cotton waste 3.4xl07 H,I Cassava peel 5.4xl07 Palm oil cheaf 3.0xl0 Vegetable
7

H,F G,F I, F,H H,F,I I,H,G

6.6xl07

Plantain leaf 3.7xl04 Sawdust 5.1xl07

Table 8. Fungal load of the sterilized substrates S/NO ANC ORGANISM IDENTIFIED H,I H,F G,F F,H,I G,F,F H,F

Cotton waste 3.4.xl07 Cassava peel 5.4xl07 Palm oil cheaf 3.0xl0 Vegetable
7

6.6xl07

Plantain leaf 3.7xl07 Sawdust 5.1xl07.

Table 9.Fungal load of the sterilized substrates S/NO ANC ORGANISM IDENTIFIED J,A,F F,H F,H,I J,H I,J,H G,F,I

Cotton waste 3.7xl06 Cassava peel 2.6xl06 Palm oil cheaf 5.2xl0 Vegetable
6 6 6

3.1xl0

Plantain leaf 4.3xl0 Sawdust

4.8xl06

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Dike K. S et al J. Microbiol. Biotech. Res., 2011, 1 (3):1-14 ______________________________________________________________________________

Table 10.Effects of pH on the substrate used Specie Pleurotus ostreatus Substrate Initial PH (before Inoculation) Sawdust 7.5 Vegetables 7.5 Dry plantain leaf 5.1 Cotton waste 4.8 Palm oil chaff 4.9 Cassava peel 7.3 Dry plantain leaf Cotton waste Palm oil cheaf Cassava peel 5.1 4.8 4.9 7.3 Final PH (After Harvest) 3.6 6.5 4.2 3.9 3.8 3.7 4.2 3.9 3.8 3.7

DISCUSSION AND CONCLUSION The agro wastes used as substrates; dry plantain leave, palm oil chaff, cassava peels cotton waste, saw dust and vegetable leaves contain lignin and cellulose which many Basidiomycetes like Pleurotus species are capable of degrading with the aid of carbohydrate and polyphenol oxides (enzymes). Lignin contains aromatic building block that are very resistant to break down. [6]. [22] noted the progressive decrease in lignin content of saw dust during the growth of Pleurotus species. They studies made by these authors have shown that soft rot Basidiomycetes such as pure placenta release hydrogen peroxide which acts as a pretreatment agent to the break down of the cellulose to simpler molecules which are utilized to growth. The spent substrate is later useful in the production of animal feed while the fruit bodies are used for human consumption because of its nutritional value and medical purposes. Microbial succession from composting to harvesting has shown that the nature and amount of nitrogen present in the mushroom substrate influences the degree of cellulose degradation. According to [7] the growth of Pleurotus species is favoured on a substrate of low nitrogen content and the nutrient content of substrates affects the formation of fruit bodies. From this work, it seems that cotton waste contains the necessary nutrients required for the fructification of Pleurotus nutrients. This is drawn from the observation that more fruit bodies were produced when cultivated on cotton waste than on vegetable leave. The substrate was capable of retaining enough water to support the growth of the mycelium up to fruit body formation. According to [12] saw dust has to be supplemented with cotton waste 5% and wheat (2 to 3%) to enrich the medium for option yield of fruit bodies. In all, the yields from all the substrates seemed to be below optimum and poor compare to saw dust. The incubation period fell within the harmattan period and its dryness adversely affected the mushroom culture. This seemed to be more manifest in sawdust which had lesser moisture during the hematite season which rapidly dried up substrates. As water was sprinted in the surface of the substrates fruit bodies were then produced. Comparing the yield of Pleurotus ostreatus (florida) on the basis of weight of their fruit bodies it was observed that vegetable leave produced fairly good yield of fruit bodies, as shown in Table 2. Generally cotton waste produced heavier fruit bodies then saw dust. One fruit body produced from vegetable leave weight whereas 12 Available online at www.scholarsresearchlibrary.com

Dike K. S et al J. Microbiol. Biotech. Res., 2011, 1 (3):1-14 ______________________________________________________________________________ the fruit bodies from saw dust the former was suspension to the letter as mushroom substrate both in terms of number and weight. [23] stated that mushroom yield declines with the suggesting depletion of nutrients from substrate. Yield increase that was observed in this work during the first flush; number of fruit bodies produced was higher than the number produced in the seed and subsequent flush (as some have was up to six flush). Studies made by [11] have shown that the fruiting for Pleurotus ostreatus was between 1-200 g, as it was observed in table 4. During the course of this work, daily temperature readings were noted and it was observed that the early morning temperature between 20-26C favoured the growth of the fungus. The decline in pH may be attributed to organic acid production in the course of solid state fermentation of the substrate. In mushroom growing, processed agricultural or industrial wastes are transformed into edible biomass and subsequently into a soil conditioner. Mushrooms are saprophytes or decomposers of organic matter, they therefore convert waste organic matter into food which without further processing is directly edible by humans. They can convert waste materials into human food. Most agricultural wastes use in the cultivation of mushroom is resistant to degradation because they contain cellulose, hemicellulose and lignin. Fungal mycelium excretes extensive enzymes complexes which can directly attack and degrade these components. They therefore use these wastes as nutrients for their growth. Mushroom growing, as an agro-industry should be encouraged in Nigeria, since the use of agricultural waste product in producing edible mushroom would improve the diet of an average Nigeria. It is also of much economic importance in not only enhancing the income generation of farmers but also saving foreign exchange by reducing importation of exotic mushrooms and replacing some components in live stock feed manufacture. It also recycles agro-waste which would have otherwise accumulated beyond tolerable limits especially as the spent substrate is reintegrated into the soil to improve its fertility and structure. Pleurotus species should be recommended for cultivation in Nigeria as there are enough agricultural wastes such as groundnut pods, rice straw, rice bran and husk, saw dust, etc. for its cultivation. This will also control environmental pollution resulting from burning of these wastes. Secondly, the average temperature of Nigeria is ideal for Pleurotus species which grows at optimum temperature of 20 to 30C. Mushroom farming is considered to be very interesting and rewarding. It is increasingly becoming a fascinating hobby, particularly for retired people and house wives who can grow mushrooms in small boxes owing to non availability of land and thus increase their income as well. REFERENCES [1] A.O.A.C, 1980, Official methods of Analysis,15th edn.Washington,D.C.Association of Official Analytical chemists.1SBN 2-93558442-0 [2] C H Anyankorah, 2002 , Cultivation of Edible mushroom. 30:399-401. 62:415. [3] N Bahl, 1985, Hand book on mushrooms. 2nd ed.oxford 85 IB Publishing Co.PVT. New Delhi. 13 Available online at www.scholarsresearchlibrary.com

Dike K. S et al J. Microbiol. Biotech. Res., 2011, 1 (3):1-14 ______________________________________________________________________________ [4] Banjo, 1998, Cultivation of Edible Mushroom The newletter of Industrial Microbiologist of Nigeria, 4:3 [5] Barnett H.I and B.B Hunter, 1985, Illustrated General of imperfect fungi.5th ed. Burges Publishing New York [6] S T Chang, 1980, J. Mushroom as human food. Bioscience [7] Chapman and Hall, London, United Kingdom. Pp.71 [8] Cooner Deanna, 2001 Mushroom farming Adventures, June-July P.14-15 [9] Elliott, C.E. (1991). Reproduction In Fungi, first edition. [10] W D Gray, 1973, The use of fungi as food processing. Journal of industrial Microbiology, 2:3-4 [11] CS James, 1995. Analytical Chemistry of Foods 1st edn, Chapman and Hall,New York [12] O Kandler, 1983, Carbohydrate metabolism in lactic acid bacteria.Antonie van Leeuwenhoek 49:209-224 [13] P Oei, 1991, Manual on Mushroom cultivation. Tool Foundation Amsterdam p.49-50 [14] P Oei, 1996, Mushroom Cultivation, First edition, supreme, Netherland pp,22-87 [15] Ogundana, S.K. and O.H Fagede, 1982, Nutritive value of some Nigerian edible mushrooms. Foods chem. 8:263-268. [16] Pelczar,M.J., Chan,E.G., and N.R Kriej, 1986, Microbiology 5th edition.McGraw Hill Book co.Singapore [17] Park, Kwnag-ho, 2001, Nutritional value of a variety of Mushroom P.5 [18] Peter, 1996 Mushroom cultivation, first edition, Pp. 1-15 [19] Quimo, T. H, Chang, S.T and J.P Roysse, 1990, Technical guidelines for mushroom growing the tropics food and Agricultural Organization of the United Nation. United Stated 1-3 [20] K Rasmussen, 1967 , Journal of Agric. Research.62:415. [21] D J Royse (ed), 1996,Mushroom Biology and Mushroom products, proceedings of the second International Conference, University Park,PA,June 9-12. [22] R H Salted, 1985, Arch. Hyg. 3:443-463. [23] RHA Smeath, 1986, Classification of Microbiolog. In assay in Microbiology 9 th edn Norris, J.r and Richman,M.H Wiley London [24] Stamets, P and Jeff Chilton, 1983, The Mushroom Cultivator Agarikon Press,Oympia WA415 Supreme, Netherland Pp. 22-87. [25] Talaro, K. P and Talaro Arthur, 2002, Foundations in Microbiology, Fourth edition.Mc Graw-Hill, New York P 687 [26] L Thomas, 1998, Mushroom, first edition. Dorling Kindersley Limited. London, United Kingdom. [27] LJ Van Griensen, 1989, The mushroom training school. Mushroom Experiment Station. Horst. The Netherlands. [28] L Zadrazil, 1976 , The ecology and industrial production of Pleurotus Ostreatus , Pleurotus Flonds , Pleurotus cornucopke . In Mushroom science ix (part I): 621-652. [29] Ztheng, Aibo (2004) Mushroom production. Odi printer, Bayelsa State, Nigeria. 2:2-12.

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