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ISSN 2278 – 0211 (Online)

Microorganisms Associated with Composting of Pycnanthus angolensis with Cow Dung

Olabode, O. O. Senior Lecturer, Integrated Science Department, Adeyemi College of Education, Ondo, Nigeria Dr. Adegunloye, D. V. Senior Lecturer, Department of Microbiology, Federal University of Technology, Akure, Nigeria Akinyele, B. J. Professor, Department of Microbiology, Federal University of Technology, Akure, Nigeria Akinyosoye, F. A. Professor, Department of Microbiology, Federal University of Technology, Akure, Nigeria

Abstract: Composting and vermicomposting processes were carried out using wood dust of Pycnanthus angolensis with animal waste added as boosters for compost. Substrates were prepared with varying ratio of wood dust: animal waste and kept for a period of 10 weeks on shelf outdoor. The temperature reached a maximum of 42.5 0C in composting in 2 weeks of the process and dropped 0 to 25.5 C in vermicomposting at the same period. At the initial stage, more of gram positive bacteria were isolated, including Bacillus coagulans, Pediococcus cerevisae, Streptococcus faecalis. Lactobacillus subger, Micrococcus luteus, Actinomyces bovis, Actinomyces ericksonii. Some of the gram negative bacteria isolated were Acinetobacter anitratis, Citrobacter freundii, Escherichia coli Pseudomonas aeruginosa and Brucella militenses. The fungi species isolated include Alternaria alternata, Aspergillus flavus, Aspergillus fumigatus, Cladosporium sp Curvularia The highest total population of bacteria was 2.55×10 7 cfu/ml, while that of fungi was 4.35 10 5 sfu/ml isolated from compost substrate of the wood dust and cow dung. The total bacteria population increased from 4.55 ×10 6 to 2.55×10 7cfu/ml in composting, and 6.82×10 6cfu/ml in vermicomposting in 2 weeks. Both bacteria and fungi diversity and population reduced significantly at the end of the process with the most occurring genus as Bacillus and Aspergillus species

Keywords: Animal waste, bacteria, decomposition, isolation, substrate

1. Introduction Composting is a natural process of recycling organic material, by the action of biodegradative microorganisms into nutrient rich product known as compost. The organic waste materials mainly of animal and plant origin are potential sources of organic matter and plant nutrient (Adeniran et al ., 2003). Animal wastes in particular are highly nitrogenous and rich in mineral elements, such as potassium, calcium and phosphorus. They are used as soil enrichment in plant cultivation. Composting is a natural biological process resulting in the controlled decay of organic matter in a warm moist environment by bacteria, fungi, and other organisms (Devi, 2012). The organic waste materials mainly of animal and plant origin are potential sources of organic matter and plant nutrient (Adeniran et al ., 2003). Animal wastes in particular are highly nitrogenous and rich in mineral elements, such as potassium, calcium and phosphorus. They are used to enrich the soil in plant cultivation. Composting is a process of solid-waste fermentation which explores the biodegradative ability of microorganisms and mineralisation. It is of importance to a mushroom grower because it affords the preparation of substrates that promote the growth of mushroom but remove pathogenic microorganisms, reduce infestation by insects, fungi, and thereby improve the yield of mushroom fruit body (Gbolagade, 2006). For a long time, composting is applied as a biological process of organic waste management in many parts of the world, but recently the use of certain species of earthworm have become of tremendous importance and advantage in vermicomposting for biological degradation of organic wastes to obtain fertilizer. The biochemical decomposition of organic matter is primarily accomplished by microorganisms, but earthworms are crucial drivers of the process as they may affect microbial decomposer activity by grazing directly on microorganisms (Aira et al., 2009; Monroy et al., 2009; Gomez-Brandon et al., 2011).

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Microorganisms which colonize mushroom compost during composting process are the crucial determinant of the nutrient status of compost that is required for mushroom cultivation. For the composting process, the importance of microbial communities is well established (Ryckeboer et al. , 2003). Previous studies have established the importance of microbial communities in the composting process. These have included bacterial population, actinobacteria and fungi (Chandna et al., 2013). Bacteria and fungi in particular, digest the complex organic compound that make up living matter and reduce them to simpler compounds that plants can use for food (Oyeyiola et al ., 2013). The biomass ratio of fungi to prokaryotes in compost is about 2:1. In addition, fungi existing in compost use many carbon sources, mainly lignocellulosic polymers and can survive in extreme conditions. They mainly are responsible for compost maturation. Fungal genera (e.g. Aspergillus, Penicillium , Fusarium, Acremonlum and Cladosporium ) are dominant in the compost process (Anastasi et al ., 2005). There are parameters that play an important role in the frequency of fungal species during a composting process including temperature, moisture, C/N ratio, pH and required oxygen to maintain the aerobic conditions. Two parameters of moisture and pH are more effective in fungal activities (Grigatti et al., 2011). Gbolagade (2006) identified eleven bacteria isolates from substrate of Terminala superba wood dust supplemented with wheat bran. The types of substrate, environmental condition would determine the type of bacteria that can colonize it. The backbone of organic agriculture is the maintenance of good soil and healthy environment through the use of compost. Cultivation of saprophytic edible mushroom may be the only currently economical biotechnology for lignocellulose organic waste recycling that combines the production of protein rich food with the reduction of environmental pollution (Obodai and Apetogbor, 2003). Mushroom is cultivated worldwide because it is able to utilise lignocellulose and grow on various agricultural wastes (Mshandete and Cuff, 2008 .). Various wastes from agricultural industry could be used to prepare compost for mushroom cultivation (Gbolagade et al ., 2011). Lignocellulosic wastes from tree wood dust are abundantly available in Nigeria for composting. Composting organic matter wastes is an important pathway for carbon flow and cycling of nutrients, both in industrial and developing countries (Bonito et al ., 2010). Bacteria associated with varying compost and vermicompost preparation using animal waste as booster has not been determined. The objective of the present study is to provide more information on the microorganisms’ types and load responsible for the composting of Pycnanthus angolensis with cow dung, for compost preparation that could be used for mushroom cultivation.

2. Materials and Methods

2.1. Collections of Animal and Agricultural Wastes Cow dung was collected inside sterile polythene bags from animal farm in Pele village located in Ondo West Local Government Area of Ondo State, Nigeria. Sawdust of Pycnanthus angolensis (Africana nut) was collected in clean bags from sawmills at Oka, Ondo where the wood was milled into 5mm sizes. Wastes were transferred to the laboratory for composting and vermicomposting preparation.

2.2. Compost Preparation The composting was prepared by passive pile method (Keith et al ., 2009). One kilogram of composting substrate was prepared by mixing wood waste of Pycnanthus angolensis and cow dung at ratios of 5:5,6:4,7:3,8:2,9:1 (numbered as 1,2,3,4 and 5) for substrates, the control sample as 100% wood dust. The various samples were composted in plastic bowls of 30cm depth and observations were made daily for 70days. The composting was kept at moisture of 65% and ambient room temperature. Temperature of the core were taken daily during the period of the experiment. Samples of the composting were taken daily for bacteriological analyses.

2.3. Vermiculture Eudrillus eugiene identified as a fast breeder and active feeder on organic matter that are high in nitrogen was used for the vermiculture (Jambhekar 1992). Earthworm culturing was done under shelter to avoid direct sunlight and heavy downpour using fifteen litre plastic buckets with perforated lid. A bed of 10cm height using sawdust as the base was sprinkled with water to get a moisture level of 40-45% which made the bed to appear wet. Different substrate preparation of Pycnanthus angolensis were mixed with the cow dung in equal quantity with appropriate quantity of water to make a homogenous mixture. The mixture was kept for two weeks, while the material was turned 2 to 3 times at 4-5 days interval. This was transferred on the layer of beddings prepared earlier. Worm was introduced into the prepared culture. The worm fed actively on the organic matter and bred (Henamgee, 2003).

2.4. Vermicomposting Compostings heaps of the various substrates were made inside plastic bowl at the rate of 10 worms/kg of feed mix (substrate). It was kept wet to a moisture level of 70% for 60 days (Henamgee, 2003). The vermicomposting formed completely gave the smell of moist soil. Samples were taken during the period of vermicomposting for 70 days for bacteria isolation and identification. Colonial characteristic of the bacterial isolates were determined using parameters such as size, elevation, pigments, surface, opacity and shape.

2.5. Substrate Samples Composted wood dust of Pycnanthus angolensis and cow dung (CPA), Pycnanthus angolensis and Vermicomposted samples of the wood dusts of Pycnanthus angolensis and cow dung were prepared like the compost (VPA), while 100% samples of wood dust without dung were used as control in composting (CPco) and vermicomposting (VPco).

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2.6. Moisture Content The moisture content of compost and vermicompost were determined by standard method of AOAC, 2005. The moisture content was determined by using oven drying method which is based on weight loss of water due to evaporation. Clean and dry dishes were weighed by using Mettler balance and their respective weights were recorded (W1). Four grams of the sample (composts and mushroom) were weighed into respective glass dishes (W2) spreading as much as possible and transferring into a desiccator immediately after each weighing, until all weighing were completed to prevent absorption of moisture from the atmosphere. The glass dishes containing the sample were transferred from desiccators into the oven maintained at 105 0c and dried in the oven until constant weights (W3) were obtained. The loss in weight during drying in % was taken to be % moisture content

2.7. Determination of the Temperature of Compost and Vermicomposts The temperatures of the samples were taken and recorded at different sampling point. A mercury-in-glass thermometer was dipped into the compost and vermicompost piles to a depth of 25cm to determine the temperature of each sample.

2.8. Isolation and Identification of Microorganisms The bacteria were isolated from the composting and vermicomposting using conventional microbiological techniques and biochemical tests according to Olutiola et al., (2000). The test carried out on the isolated bacteria were Gram staining, spore staining, motility, sugar fermentation, catalase, oxidase, coagulase, and indole tests. The sugars used for the fermentation tests were glucose, sucrose, galactose, mannitol and lactose. After Gram staining, the Gram reaction and shapes of the cells were examined under a light microscope with oil immersion objectives lens at a magnication of 100x. Auxanographic features were determined with the API 20 E System (Api Biomerieux SA, France). One gram of various samples was mixed with 9ml of sterile water to solution samples. This was diluted up to 10 -5 serial dilution using sterile pipette/one millilitre (1ml) of the solution sample. One milliliter of an appropriately diluted culture of the 10 -4 and 10 -5 were poured into plates with potato dextrose agar and Czapek agar for fungi and nutrient agar and Mackonkey agar for bacteria. Streptomycin sulphate (0.05g/dm 3) was added for fungal media to prevent bacteria contamination (Jonathan and Fasidi, 2001). The isolates were plated in triplicates and incubated for 72hrs and 24hrs at 30 20C and 37 0C for fungi and bacteria respectively until the colonies appeared. At the end of incubation period the plates were observed for fungal and bacteria growth and different colonies was sub cultured on fresh plates of culture media. The pure cultures were stored in the refrigerator as stock cultures for subsequent test. The morphological characteristics as well as the biochemical tests were done on the different colonies of bacteria while microscopic identification of the fungal isolates was done using the method of Prescott. (2008). Macroscopic examination of fungal growth was carried out by observing the colony morphology, diameter, colour (pigmentation), texture and surface appearance. Fungi were identified by observing their macroscopic characteristics (colour, texture, appearance and diameter of the colonies) and microscopic characteristics (microstructure).

2.9. Statistical Analysis of Results All data generated were analysed statistically as described by the method of Olawuyi (1993). Statistical values that were calculated include mean and standard deviation.

3. Results and Discussion The temperature varied throughout the process and reached a maximum of 42.5 0C in compost in one week of the process and dropped to at least 25.5 0C in vermicomposting at the same period. Eighteen species of bacteria and sixteen of fungi were isolated in all substrates of Pycnanthus angolensis wood dust in this study. The bacteria include, Acinetobacter anitrates, Actinomyces bovis, Lactobacillus jense, Bacillus coagulans, Enterobacter aerogenes, Micrococcus roseus, Bacillus subtilis, Clostridium perfringes, Pseudomonas aeruginosa, Bacillus cereus, Bacillus licheniformis, Escherichia coli, Pediococcus cerevisae, Lactobacillus subger, Brucella melitensis, Micrococcus luteus, Actinomyces ericksonii, Streptococcus faecalis (Table 1 and 2), and the fungi species are Alternaria alternata, Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Aspergillus ochraceous, Aspergillus versicolor, Aspergillus parasiticus, Cladosporium sp., Curvularia lunata, Fusarium oxysporum, Geotrichum sp., Mucor racemosus, Mucor mucedo, Penicillium lanosum, Penicillium nigricans, Rhizopus oryzae, Rhizopus stolonifer, Scopulariopsis sp. a nd Trichoderma sp (Table 3 and 4). The number of species reduced to 9 bacteria in composting and 10 in vermicomposting at the end of the process. Bacteria species reduced in all substrate mix at the end of composting and vermicomposting. Bacteria species and load in the control were lower than in the wood dust substrate mix with organic waste. Bacteria of the family enterobacteriaceae that were present at the early stage in all the substrates were eliminated at the end of the process in composting and vermicomposting. The total bacteria population increased in 2 weeks of process from 4.55 x 10 6 to 2.55x 10 7cfu/ml.in composting and 6.82 x 10 6cfu/ml in vermicomposting when Pycnanthus angolensis wood dust was used (Table 1 and 2). The population reduced to 1.61 x 10 6 in compost and 3.24 x 106 in vermicompost at the end of the process. INTERNATIONAL JOURNAL OF INNOVATIVE RESEARCH & DEVELOPMENT Page 178

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Enterobacter aerogenes has the highest bacteria load in composted wood dust with cow dung (experimental) CPA/VPA at the 0 day, while Pediococcus cerevisae has a least population of at the same stage. Micrococcus roseus experienced the highest growth in two weeks of composting and of vermicompost on the same substrates. The highest total bacteria count was observed in composting wood dust in two weeks, while the least population was isolated from control sample (CPco) in 10 weeks of composting. The highest bacteria load was isolated in composted wood dust with cow dung (experimental) CPA/VPA at 0day, while Pediococcus cerevisae has a least population the same stage. Micrococcus roseus experienced the highest growth in two weeks of composting and vermicomposting on the same substrates. The highest total bacteria count was observed in composting wood dust in two weeks, while the least was isolated from control sample (CPco) in 10 weeks of composting. The result showed variation in bacterial species (Table 1 and 2) in composting and vermicomposting of wood dust and organic wastes used in this experiment. Aspergillus fumigatus has the highest fungal load in vermicomposted wood dust with cow dung (experimental) CPA/VPA at the final stage, while Aspergillus flavus has a least population at the same stage in the compost. Aspergillus fumigatus experienced the highest growth in 10weeks of composting and of vermicomposting on the same substrate. The highest total fungal count of 4.35 x 10 5sfu/ml was observed in vermicomposted wood dust at the end. The result also showed variation in fungi species (Table 3 and 4) in composting and vermicomposting of wood dust and organic wastes used in this experiment. These further confirm that the wood dust and various wastes used to prepare the compost are heavily laden with bacterial and fungal population. For the composting process, the importance of microbial communities is well established (Ryokeboer et al., 2003). Bacteria and fungi in particular, digest the complex organic compound that make up living matter and reduce them to simpler compounds that plants can use for food (Oyeyiola et al ., 2013). Studies on bacterial population, actinobacteria and fungi during composting have been reported extensively (Sundberg et al., 2011). The abundance of distribution of bacterial population and genus in this work is supported by the work of Gbolagade (2006) who identified eleven bacteria species isolated from substrate of Terminala superba wood dust supplemented with wheat bran. Adegunloye et al ., (2007) isolated seven species in the work using compost of agricultural wastes with cow dung as booster. In both works, Bacillus was the most frequently isolated (Figure 1). Isolation of aerobic bacteria is favoured by the turning of compost heap for oxygen to have its free flow and support for life. Most of the bacteria isolated from the compost and vermicompost are those that are able to grow in mesophilic environment. The abundance of distribution of fungi population and genus in this work is supported by that of Anastasia et al ., (2005) who isolated a total of 194 fungal entities from compost and vermicompost, which include fungal genera Fusarium, Cladosporium, Penicillium , Rhizopus and related species of Aspergillus . He also identified A. fumigatus . Ribollido et al ., (2008) in his study of municipal waste isolated Penicillium, Alternaria, Aspergillus Trichoderma Ulocladium though Ulocladium was not isolated in this work. Adegunloye et al ., (2007) identified Aspergillus species as among the predominant fungi in compost of agricultural wastes using cow dung as booster. The results of this study revealed that one of the most frequent and medically important fungi A. Fumigatus is one of the problems that occur during the compost process by emitting of bio aerosols which can contaminate the areas surrounding the compost especially while turning compost pile for a eration (Nadal, et al ., 2009). Moreover, A. Fumigates may cause serious allergic diseases in people who are genetically predisposed to these diseases (Browny et al .,2001).

Figure 1: Effects of period of composting on temperature of Figure 2: Effects of period of vermicomposting on P.angolensis wood dust and cow dung (CPA). Ratio of wood temperature of P.angolensis wood dust and cow dung (VPA). dust: cow dung Ratio of wood dust: cow dung

The highest bacterial species isolated throughout the composting process is Bacillus. Bacillus polymyxa and Bacillus licheniformis are the most occurring of this group. The total bacteria load was high in both composting and vermicomposting of wood dust and organic waste. It increased in composting and in vermicomposting. There are more of bacteria species in the vermicomposting than the composting at the end of the process. The total bacterial counts of the composting and vermicomposting samples ranged between of 8.12 x 10 5cfu/g in control sample (wood dust without organic matter) at the end of the experiment and the highest of 2.55x10 7cfu/g in wood dust and cow dung in 2weeks of the process. The total bacteria population in vermicompostimg was higher than the population in composting at the end of the experiment. This may be caused by the activity of earthworm during vermicomposting. Earthworm activity decreased the population of coliform significantly at the end of vermicomposting (Table 3). Passage through the guts of Eudrillus eugeniae must have accounted for this. Monroy et al, (2009) observed that the process through the guts of Eisenia andrei,

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Eisenia fetida and Eudrillus eugeniae reduced the population of total coliforms by 98% relative to that of fresh pig slurry. Kumar found the amount of Escherichia coli O157:H7 reduced through digestion of the earthworm (Kumar and Sekaran, 2005). Liu et al, (2011) isolated 6 Lactobacillus strains from vermicompost of Eisenia fetida , which was stressed by Escherichia coli O157:H7 and each strain obviously could restrict the pathogen obviously. Animal dungs are rich in microbes in which bacteria constitute the largest fraction and dominate the first stage of decomposition, making waste available for earthworm, hence affecting the growth of coliform, but fungi may be present as spores. Animal dungs are rich in microbes in which bacteria constitute the largest fraction and dominate the first stage of decomposition, making waste available for earthworm, hence affecting the growth of coliform, but fungi may be present as spores. The most frequently occurring specie in all the substrates is Micrococcus luteus and Pseudomonas aeruginosa (Figure 3). This frequency of isolation of Micrococcus luteus and Pseudomonas aeruginosa can be attributed to their ability to grow in mesophilic environment and to colonise vast diversity of habitat, which was most prevalent during the process of both composting and vermicomposting (Adegunloye et al ; 2007). They commonly inhabit soil and fresh water. Micrococcus luteus are frequently found on skin of animals. Species like Clostridium was observed to be present in the compost and vermicompost throughout the process. These can be due to the ability to survive high temperature habitat that was prevalent at the earlier stage. They are able to produce heat resistant spores. Other facultative anaerobes present in the compost substrates are Micrococcus roseus and Citrobacter freundii . They are found in water faeces and urine. They are normal intestinal inhabitants

Bacteria Species C (0 day) C(2wks) C(10wks) VC(0 day) VC(2wks) VC(10wks) Acitnomyces bovis 6.0 x 10 5 2.8 x 10 4 - 6.0 x 10 5 - - Pseudomonas 1.7 x 10 5 3.5 x 10 5 2.5 x10 5 1.7 x 10 5 4.7 x 10 5 4.5 x 10 5 aeruginosa Lactobacillus jense 3.2 x 10 5 8.2 x 10 6 4.1 x 10 4 3.2 x 10 5 7.8 x 10 5 2.8 x 10 5 Bacillus coagulans 4.3 x 10 5 8.1 x 10 5 - 4.3 x 10 5 7.3 x 10 5 - Pediococus cerevisae 2.0 x 10 4 - 6.4 x 10 5 2.0 x 10 4 - 5.2 x 10 5 Lactobacillus subger 7.0 x 10 4 5.6 x 10 6 2.4 x 10 3 7.0 x 10 4 7.2 x 10 5 4.3 x 10 4 Brucella melitenses - 3.4 x 10 4 9.3 x 10 4 - - 8.2 x 10 5 Micrococcus luteus 4.2 x 10 4 7.1 x 10 5 7.2 x 10 4 4.2 x 10 4 8.2 x 10 5 - Actinomyces eriksonii 5.2 x 10 5 2.9x 10 4 - 5.2 x 10 5 1.7 x 10 5 2.1 x 10 4 Bacillus licheniformis 2.5 x 10 5 7.8 x 10 5 - 2.5 x 10 5 7.5 x 10 5 - Enterobacter aerogenes 6.7 x 10 5 2.1± x 10 5 - 6.7 x 10 5 3.0 x 10 4 - Micrococcus roseus 5.3 x 10 4 6.1 x 10 6 2.7 x 10 5 5.3 x 10 4 7.2 x 10 5 7.1 x 10 5 Bacillus subtilis 4.3 x 10 5 6.8 x 10 5 2.7 x 10 5 4.3 x 10 5 7.3 x 10 5 7.6 x 10 4 Clostridium perfringes 1.5 x 10 5 1.2x 10 6 1.9 x 10 4 1.5 x 10 5 8.5 x 10 5 2.7 x 10 5 Bacillus cereus 2.7 x 10 5 7.5 x 10 5 - 2.7 x 10 5 8.2 x 10 5 5.2 x 10 4 Escherichia coli 5.5 x 10 5 4.7 x 10 4 - 5.5 x 10 5 4.5 x 10 4 - Total 4.55± 0.25 x 2.55±0.20 1.61±0.15 x 4.55±0.40 x 6.82± 0.20x 3.24± 0.45x 10 6 x10 7 10 6 10 6 10 6 10 6 Table 1: Bacteria load (CFU/g) from composting (C) and vermicomposting (VC) of P. angolensis and cow dung Key: - = Absent

Bacteria Species C (0 day) C(2wks) C(10wks) VC(0 day) VC(2wks) VC(10wks) Acinetobacter anitratis 5.0 x10 5 8.5 x 10 5 2.5 x 10 5 5.0 x 10 5 5.5 x 10 5 4.1 x 10 5 Pseudomonas aeruginosa - 3.5 x 10 5 4.3 x 10 5 - 3.0 x 10 5 4.0 x 10 5 Pediococus cerevisae 3.0 x10 4 - - 3.0 x 10 4 1.7 x 10 4 - Brucella melitenses 5.0 x10 4 4.4 x 10 4 1.2 x 10 4 5.0 x 10 4 3.0 x 10 4 - Micrococcus luteus 3.0 x10 5 1.5 x 10 5 1.2 x 10 4 3.0 x 10 5 6.0 x 10 4 2.0 x 10 4 Bacillus polymyxa 3.0 x10 5 4.0 x 10 5 - 3.0 x 10 5 2.0 x 10 5 1.5 x 10 4 Bacillus licheniformis 1.9 x10 4 1.8 x 10 4 - 1.9 x 10 4 2.0 x 10 5 - Clostridium perfringes 3.0 x 10 4 3.5 x 10 5 - 3.0 x 10 4 3.0 x 10 5 - Total 1.23±0.10 2.16±0.30 x 8.12±0.15 x 1.23±0.40 x 1.66±0.25 x 8.45±0.50 x x 10 6 10 6 10 5 10 6 10 6 10 5 Table 2: Bacteria load (CFU/g) from composting (C) and vermicomposting (VC) of P. angolensis Key: - = Absent

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Fungal Specie C(0 day) C(2wks) C(10wks) VC(0 day) VC(2wks) VC(10wks) Alternaria alternata 4.8 x 10 3 4.2 x 10 3 2.1 x 10 3 4.8 x 10 3 5.2 x 10 3 2.9 x 10 3 Aspergillus flavus 2.5 x 10 3 2.2 x 10 3 1.3 x 10 3 2.5 x 10 3 1.2 x 10 3 Aspergillus fumigatus 3.1 x 10 3 4.7 x 10 3 6.1 x 10 4 3.1 x 10 3 3.4 x 10 3 3.5 x 10 5 Aspergillus niger 9.3 x 10 3 8.2 x 10 3 2.0 x 10 4 9.3 x 10 3 4.6 x 10 3 1.0 x 10 4 Aspergillus versicolor 2.7 x 10 3 4.2 x 10 4 2.7 x 10 4 2.7 x 10 3 5.3 x 10 3 5.9 x 10 3 Aspergillus parasiticus 7.2 x 10 3 7.1 x 10 3 4.2 x 10 4 7.2 x 10 3 2.5 x 10 3 1.9 x 10 3 Cladosporium sp. 9.8 x 10 3 1.5 x 10 4 4.2 x 10 4 9.8 x 10 3 1.8 x 10 4 5.0 x 10 4 Fusarium oxysporum 4.1 x 10 3 7.3 x 10 3 2.8 x 10 4 4.1 x 10 3 2.2 x 10 3 Mucor racemosus 5.1 x 10 3 6.9 x 10 3 2.7 x 10 3 5.1 x 10 3 3.2 x 10 3 1.5 x 10 3 Penicillium lanosum 4.1 x 10 3 8.4 x 10 3 2.5 x 10 3 4.1 x 10 3 1.8 x 10 4 4.9 x 10 3 Penicillium nigricans 4.5 x 10 3 7.9 x 10 3 4.2 x 10 3 4.5 x 10 3 2.3 x 10 3 Rhizopus oryzae 3.2 x 10 3 3.3 x 10 3 1.3 x 10 3 3.2 x 10 3 2.2 x 10 3 Rhizopus stolonifer 3.2 x 10 3 4.3 x 10 3 6.9 x 10 3 3.2 x 10 3 2.5 x 10 3 Scopulariopsis sp. 5.2 x 10 3 3.5 x 10 3 1.4 x 10 3 5.2 x 10 3 6.6 x 10 3 2.2 x 10 3 Trichoderma sp. 7.6 x 10 3 6.4 x 10 3 1.7 x 10 4 7.6 x 10 3 5.8 x 10 3 4.8 x 10 3 Total 7.64±0.20 x 1.31±0.15 x 2.59±0.40 x 7.64±0.30x 8.18±0.40 x 4.35±0.20 x 10 4 10 5 10 5 10 4 10 4 10 5 Table 3: Fungi load (SFU/g) from composting (C) and vermicomposting (VC) on wood dust of P. angolensis and cow dung. Key: - = Absent

Fungal Specie C(0 day) C(2wks) C(10wks) VC(0 day) VC(2wks) VC(10wks) Aspergillus flavus 3.8 x 10 3 4.8 x 10 3 3.9 x 10 3 3.8 x 10 3 4.7 x 10 3 2.4 x 10 3 Aspergillus fumigatus 2.1 x 10 3 3.7 x 10 3 3.6 x 10 3 2.1 x 10 3 6.8 x 10 3 3.9 x 10 3 Aspergillus niger 4.2 x 10 3 8.3 x 10 3 2.8 x 10 3 4.2 x 10 3 6.3 x10 3 3.3 x 10 3 Aspergillus ochraceous - - 2.6 x 10 3 - 1.6 x 10 3 - Fusarium oxysporum 2.5 x 10 3 3.2 x 10 3 1.9 x 10 3 2.5 x 10 3 3.8 x 10 3 2.6 x 10 1 Penicillium lanosum - 2.8 x 10 3 - - 3.6 x 10 3 2.1 x 10 3 Rhizopus oryzae 1.9 x 10 3 3.4 x 10 3 2.8 x 10 3 1.9 x 10 3 2.8 x 10 3 - Scopulariopsis sp. 3.2 x 10 3 1.9 x 10 3 - 3.2 x 10 3 1.8 x 10 3 2.3 x 10 3 Total 1.77± 0.10 x 2.81±0.25 x 1.76±0.35 x 1.77± 0.15x 3.14±040. x 1.43± 0.20 x 10 4 10 4 10 4 10 4 10 4 10 4 Table 4: Fungi load (SFU/g) from composting (C) and vermicomposting (VC) on P. angolensis (100%). Key: - = Absent

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Figure 3: Frequency distribution of (a) bacteria and (b) fungi species in terms of the period of sampling of the compost and vermicompost of Pycnanthus angolensis

Composting as a process is affected by various factors ranging from biology to environment. The quality of compost is dependent on variation of these factors. The composition of the substrates used provided the organic matter base of high carbon and nitrogen, and also the organisms for the decomposition process. The various minerals present in the organic matter provide a good nutrient support for the growth of microorganisms. Animal wastes in particular are highly nitrogenous and rich in mineral elements such as potassium, calcium and phosphorus. They are used as soil enrichment in plant cultivation. Adeniran et al ., 2003 stated that the organic waste materials mainly of animal and plant origin are potential sources of organic matter and plant nutrient. The result in table 3 showed that there was increase in bacteria population which indicate growth, that was initiated immediately the moisture content was increased by the addition of water to 65% and organic waste which was mixed with the wood dust. The result showed that growth was highest in the first 2weeks of the experiment which indicate the fresh presence of nutrients and bacteria that are able to initiate decomposition. Gbolagade (2006) reported that there was highest increase in microbial population in the early stages of composting which was dependent on initial substrate used and environmental condition of the composting. Chandna et al., 2013 stated that high content of degradable organic compound in the initial mixture might have stimulated microbial growth involved in self-heating during the initial stages of composting. The result in this work is similar to the observed by Devi et al., (2012) when handling poultry and paddy wastes in composting; their bacterial population was maximum at 2nd week of process. He attributed the increase to the availability of easily utilizable substrate for bacteria from fungal degradation of the composting material. The result shows variation in bacterial species in composts and vermicomposts of wood dust and organic wastes used in this experiment. These further confirm that the wood dust and various wastes used to prepare the compost are heavily laden with bacterial population. Isolation of aerobic bacteria is favoured by the turning of compost heap for oxygen to have its free flow and support for life. Most of the bacteria isolated from the composting and vermicomposting are those that are able to grow in mesophilic environment. There was difference in mineral content and quantities of compost and vermicompost. The quantity of Nitrogen was highest in compost and vermicompost with 7.72± 0.01 and 6.99± 0.41 respectively in substrate ratio 5:5, while the least was in the control. This could be as a result of bacteria and fungi contained in the animal waste in substrate of ratio 5:5(wood dust: animal waste), which was higher in any substrate mixes. It was observed that the compost wood dust and animal waste produced more minerals than the control which was 100% wood dusts. More Iron was observed in vermicompost than composts. Microbes in the gut of earthworms might have played important role in increasing the rate of mineral weathering which may account for more of Phosphorus, Calcium and Copper. Potassium was expected to be higher in vermicompost than compost. The lower content can be attributed to absence of silicate which is needed for the non - exchangeable K pool which passes through the gut of worms that are present in soil.

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Ra Mg N % P % K % Na % Ca % Cu mg/kg Zn mg/kg Mn mg/kg Fe mg/kg Ratios mg/kg Substrates 5:5 Compost 7.72±0.01 0.79±0.01 1.43±0.01 0.38±0.01 1.52±0.01 1.00±0.01 6.42±0.01 63.26±0.01 34.22±0.02 97.57±0.01 Vermicompost 6.99±0.41 1.26±0.55 0.72±0.23 2.00±0.38 12.83±0.22 0.34±0.03 29.65±0.05 54.98±0.23 39.05±0.51 785.79±0.33

1.05 6:4 Compost 6.10±0.01 0.79±0.02 0.40±0.01 1.50±0.01 1.10±0.00 7.24±0.02 59.28±0.03 33.06±0.01 239.56±0.01 ±0.01 Vermicompost 6.61±0.41 1.87±0.31 0.68±0.20 1.79±0.24 12.17±0.52 0.33±0.02 29.59±0.55 15.27±0.26 33.16±0.30 68.11±0.56

7:3 Compost 5.88±0.01 0.67±0.01 1.44±0.01 0.41±0.01 1.42±0.01 1.10±0.00 7.66±0.01 49.52±0.01 32.63±0.03 325.24±0.01 Vermicompost 7.86±0.21 0.99±0.57 0.57±0.23 1.13±0.52 2.24±0.31 0.33±0.02 32.98±0.27 34.40±0.05 29.91±0.19 562.92±0.29

1.35 8:2 Compost 5.86±0.01 0.65±0.01 0.42±0.01 1.52±0.02 1.19±0.00 8.42±0.01 42.44±0.01 28.42±0.02 290.66±0.01 ±0.01 Vermicompost 6.66±0.00 2.45±0.54 0.53±0.23 0.8±0.48 12.91±0.29 1.01±0.55 30.99±0.58 13.71±0.56 38.53±0.48 405.92±0.36

9:1 Compost 4.76±0.01 0.53±0.01 0.52±0.01 0.50±0.01 2.52±0.01 1.08±0.00 8.22±0.01 39.57±0.01 41.06±0.01 231.18±0.20 Vermicompost 5.47±0.79 0.30±0.03 0.51±0.13 0.70±0.21 12.28±0.41 0.40±0.04 32.81±0.06 37.43±0.58 21.64±0.43 279.07±0.30

Co Compost 4.04±0.01 0.59±0.03 0.49±0.03 0.41±0.02 0.88±0.01 0.08±0.002 4.79±0.02 44.27±0.02 30.27±0.05 96.23±0.03 Vermicompost 1.88±0,03 0.21±0.02 0.58±0.01 0.88±0.01 6.51±0.04 0.27±0.02 18.10±0.00 20.41±0.05 12.31±0.02 345.43±0.02 Table 5: Mineral composition of Compost and Vermicompost (P. angolensis wood dust and cow dung) Values are mean of triplicate measurements ± Standard Error of Mean (SEM) Key: Ratio - wood dust: animal waste Co -100% wood dust

4. Conclusions The main purpose of the composting for a mushroom grower is to prepare a substrate in which the growth of mushroom is promoted. The high quantity of both macro and micro elements present in the compost and vermicompost product expressed the biodegrative ability of the various bacteria and fungi present during the process. Enumeration of bacteria and fungi associated with compost was necessary in order to know the microorganisms that are responsible for optimal compost quality for maximal Pleurotus yield. The work has enabled to us to find out the presence of microorganisms harmful to man and animal. According to the results of this study, pathogenic bacterial and fungal species, and saprophytes were identified during the compost process. Composting and vermicomposting make the substrate safer to handle by reducing the microbial load, especially of enteric bacteria as seen in this work. One of the main problems of producing compost is the growth of fungal pathogens during the compost process Considering that some of these fungal species such as A.fumigatus are human pathogens and can cause serious diseases in those who are involved in the production of compost, preventive measures must be provided, and should be taken by the workers involved in the compost production, in order to prevent inhalation and infection of spores. Regarding the importance of the composting sites, finding suitable location for producing compost and also efficient management during the composting process can be effective to improve the individual health and hygienic conditions of the composting sites. The compost and vermicompost produced are expected to be used for the cultivation of Pleurotus ostreatus , to determine the effect of the process on the yield of the mushroom. Further work can also be carried out to determine the diversities of microorganisms present in the spent substrate at the end of mushroom cultivation.

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