Isolation, characerization and antimicrobial susceptibility test of soil microorganisms isolated within the cassava mill industry by BAMIGBOYE, Olayemi J.docx

CHAPTER ONE1.0INTRODUCTIONCassava (Manihot esculenta Crantz, synonymous with Manihot utilissima Rhol) belongs to the family Euphorbiaceae. It is mainly a food crop whose tubers are harvested between 7-13 months based on the cultivars planted (Cook, 1985; Taye, 1994). Cassava (Manihot esculenta Crantz) is primarily grown for its starch containing tuberous roots, which are the major source of dietary energy for more than 500 million people in the tropics (Lyman, 1993). The ability of cassava to grow and produce relatively well in marginal environment under low management levels makes it an attractive crop for poor resource (Bencini, 1991). As a food crop, cassava fits well into the farming systems of the small holder farmers in Nigeria because it is available year round, thus providing household food security. Cassava tubers can be kept in the ground prior to harvesting for up to two years, but once harvested, they begin to deteriorate. To forestall early deterioration, and also due to its bulky nature, cassava is usually traded in some processed form. The bulky roots contain much moisture (60 65%), making their transportation from rural areas difficult and expensive. Processing the tubers into a dry form reduces the moisture content and converts it into a more durable and stable product with less volume, which makes it more transportable (IITA, 1990; Ugwu, 1996). Over the years, cassava has been transformed into a number of products both for domestic (depending on local customs and preferences) and industrial uses. Cassava in the fresh form contains cyanide, which is extremely toxic to humans and animals; there is therefore a need to process it to reduce the cyanide content to safe levels (Eggelston et al., 1992).

The poor post harvest storage life of fresh cassava tubers is a major economic constraint in its utilization (Kehinde, 2006).Cassava processing generates solid and liquid residues that are hazardous in the environment (Cumbana et al., 2007; Jyothi et al., 2005). On the average, 2.62 m3 ton-1 of residues from washing and 3.68 m3 ton-1 from the water residues of flour production (Horsfall et al., 2006 and Isabirye et al., 2007). There are two important biological wastes derived from cassava processing which are the cassava peels and the liquid squeezed out of the fermented parenchyma mash (Oboh, 2006). Cassava effluents are liquid wastes from the cassava mill which are usually discharged on land or water in an unplanned manner. The cassava peels derived from its processing are normally discharged as wastes and allowed to rot in the open with a small portion used as animal feed, thus resulting in health and environmental hazards. (Desse and Taye, 2001; Aderiye and Laleye, 2003) the edible tubers are processed into various forms which include chips, pellets, cakes and flour. The flour could be fried to produce gari or steeped in water to ferment to produce fufu when cooked (Oyewole and Odunfa, 1992). The production and consequent consumption of cassava have increased extensively in recent times. This increased utilization of processed cassava products has equally increased the environmental pollution associated with the disposal of the effluents (Akani et al., 2006; Adewoye et al., 2005)In most areas, cassava mills are mainly on small scale basis, owned and managed by individuals who have no basic knowledge of environmental protection. Though on small scale basis, there are many of them, which when put together, create enormous impact on the environment.Oboh, 2005 identified two important wastes that are generated during the processing of cassava tubers to include cassava peels and liquid squeezed out of the mash. The bioconversion of the cassava wastes have been documented (Antia and Mbongo, 1994; Okafor, 1998; Raimbault, 1998; Twenyongyere and Katongole, 2002; Oboh, 2005). The wastewater contains heavy loads of microorganisms, lactic acid, lysine, amylase capable of hydrolyzing the glycosides (Raimbault, 1998; Akindahunsi et al., 1999).During the processing of cassava tubers in various products, liquid wastewaters generated was reported to cause serious havoc to vegetation, houses and bring about infection. The liquid squeezed out can be dried and used as animal feeds (Okafor, 1998; Oboh and Akindahunsi, 2003a).Microorganisms are very important members of the soil ecosystem. They play significant roles in the various transformations that go on in the soil. An important function of soil organisms is the decomposition of organic residues. This decomposition process is driven by decomposer organisms which consist of a community of soil biota including microflora and soil fauna (Swift et al., 1979; Tian et al., 1995). Fungi and bacteria are responsible for the biochemical processes in the decomposition of organic residues (Anderson and Ineson, 1983; Dinda, 1978).Despite their importance in soil, the relative abundance and distribution of these soil organisms is determined by several environmental factors. The soil is the final recipient of all forms of environmental pollutants and of recent such pollutants have had significant effects on soil microbial populations (Ogboghodo et al., 2001). Various studies of the microbiology of hydrocation degradation in soil indicate the presence of microflora in the soil that is able to degrade a wide variety of hydrocarbons (Niessen, 1970). Soon after the widespread use of antibiotics began in the early 1950s it became apparent that strains of bacteria were becoming resistant to specific antibiotics, it was later discovered in Europe that enterobacteriaceae can transfer multiple resistances from one organism to another and even from one specie to another by means of an extra chromosomal hereditary factors. The problem of antibiotic resistance could be attributed to long time use of a particular antibiotic by humans, this then makes bacteria to adapt very well with whatever challenges the antibiotics might pose and subsequently it will become resistant to such antibiotics.1.1STATEMENT OF THE PROBLEMCassava is important to human because they serve as a source of staple food for him and his animals, it could be employed to produce chips, gari, fufu etc. it is also a source of viable income for farmers who plant it and also to people involved in turning it into finished product e.g. the gari producers, the transport drivers. But as beneficial as cassava is to humans, its effluent has always constituted a source of nuisance as well as the odour emanating from gari processing plants are always offensive.Due to effluents produced in the environment, studies have been done to find out the microorganism that can be either pathogenic or non-pathogenic that are present within the cassava mill factories

1.2AIMS AND OBJECTIVESTo isolate, characterize and carry out antimicrobial susceptibility test on soil microorganisms present within the cassava mill industry.

CHAPTER TWO2.1LITERATURE REVIEWCassava (Manihot esculenta Crantz, synonymous with Manihot utilissima Rhol) belongs to the family Euphorbiaceae. It is mainly a food crop whose tubers are harvested between 7-13 months based on the cultivars planted (Cook, 1985; Taye, 1994). The tubers are quite rich in carbohydrates (85-90%) with very small amount of protein (1.3%) in addition to cyanogenic gloucoside (Linamarin and Lotaustiallin). (Nwabueze and Odunsi, 2007; Oyewole and Afolami, 2001). This high carbohydrate content makes cassava a major food item especially for the low income earners in most tropical countries especially Africa and Asia (Desse and Taye, 2001; Aderiye and Laleye, 2003).The edible tubers are processed into various forms which include chips, pellets, cakes and flour. The flour could be fried to produce gari or steeped in water to ferment to produce fufu when cooked (Oyewole and Odunfa, 1992).Fermentation is one of the oldest and most important traditional food processing and preservation techniques. Food fermentations involve the use of microorganisms and enzymes for the production of foods with distinct quality attributes that are quite different from the original agricultural raw material. The conversion of cassava (Manihot esculenta, Crantz syn. Manihot utilissima Pohl) to gari illustrates the importance of traditional fermentations. Cassava is native to South America but was introduced to West Africa in the late 16th century where it is now an important staple in Nigeria, Ghana, Ivory Coast, Sierra Leone, Liberia, Guinea, Senegal and Cameroon. Nigeria is one of the leading producers of cassava in the world with an annual production of 35-40 million metric tons. Over 40 varieties of cassava are grown in Nigeria and cassava is the most important dietary staple in the country accounting for over 20% of all food crops consumed in Nigeria. Cassava tubers are rich in starch (20-30%) and, with the possible exception of sugar cane; cassava is considered the highest producer of carbohydrates among crop plants.Despite its vast potential, the presence of two cyanogenic glucosides, Linamarin (accounting for 93% of the total content) and lotaustralin or methyl Linamarin, that on hydrolysis by the enzyme linamarase release toxic HCN, is the most important problem limiting cassava utilization. Generally cassava contains 10-500 mg HCN/kg of root depending on the variety, although much higher levels, exceeding 1000 mg HCN/kg may be present in unusual cases. Cassava varieties are frequently described as sweet or bitter. Sweet cassava varieties are low in cyanogens with most of the cyanogens present in the peels. Bitter cassava varieties are high in cyanogens that tend to be evenly distributed throughout the roots. Environmental (soil, moisture, temperature) and other factors also influence the cyanide content of cassava (Bokanga et al; 1994). Low rainfall or drought increases cyanide levels in cassava roots due to water stress on the plant. Apart from acute toxicity that may result in death, consumption of sub-lethal doses of cyanide from cassava products over long periods of time results in chronic cyanide toxicity that increases the prevalence of goiter and cretinism in iodine-deficient areas. Symptoms of cyanide poisoning from consumption of cassava with high levels of cyanogens include vomiting, stomach pains, dizziness, headache, weakness and diarrhea (Akintonwa et al; 1994).Chronic cyanide toxicity is also associated with several pathological conditions including konzo, an irreversible paralysis of the legs reported in eastern, central and southern Africa (Howlett and Konzo, 1994), and tropical ataxic neuropathy, reported in West Africa, characterized by lesions of the skin, mucous membranes, optic and auditory nerves, spinal cord and peripheral nerves and other symptoms (Oshuntokun, 1994). Without the benefits of modern science, a process for detoxifying cassava roots by converting potentially toxic roots into gari was developed, presumably empirically, in West Africa. The process involves fermenting cassava pulp from peeled, grated roots in cloth bags and after dewatering, the mash is sifted and fried.Microbial fermentations have traditionally played important roles in food processing for thousands of years. Most marketed cassava products like gari, fufu, pupuru, apu etc., in Africa are obtained through fermentation. The importance of fermentation in cassava processing is based on its ability to reduce the cyanogenic glucosides to relatively insignificant levels. Unlike alcoholic fermentation, the biochemistry and microbiology is only superficially understood, but it is believed that some cyanidrophilic/cyanide tolerant microorganisms effect breakdown of the cyanogenicglucoside. It has been shown that the higher the retention of starch in grated cassava the better the detoxification process. This could be attributed to the fermentative substrate provided by the starch. Also, the longer the fermentation process the lower the residual cyanide content.Generally, fermented cassava products store better and often are low in residual cyanide content. (Onabowale, 1988) developed a combined acid hydrolysis and fermentation process at FIIRO (Federal Institute for Industrial Research, Oshodi, Nigeria) and achieved a 98% (approx.) reduction in total cyanide after dehydration of the cassava flour for use in the feeding of chickens. Cassava roots can be industrially applied for obtaining starch and flour. However, cassava industries generate some undesirable sub-products, such as solid residues and a liquid effluent named manipueira, which may represent a major disposal problem due to the high organic charge and toxic potential, resulting from the presence of cyanoglucosides. Manipueira is rich in potassium, nitrogen, magnesium, phosphorous, calcium, sulfur and iron, presenting a great potential as an agronomic fertilizer. It contains cyanoglucosides, which explains the application as nematicide and insecticide (Palmisano et al., 2001).Cyanoglucosides are secondary metabolites produced by several plant species (Conn, 1994) used in animal and human diets, such as: apple, bamboo shoot, cassava, cherry, lima bean, maize, oat, peach, papaya, sorghum and wheat (Muro, 1989). These compounds are dispersed throughout the plant organs, mostly in non-edible parts (Jones, 1998), but may become concentrated in edible roots and leaves, as in the case of cassava. Cassava (Manihot esculenta Crantz) roots and leaves contain high concentrations of Linamarin (alpha-hydroxyisobutyronitrile-beta-D-glucopyranoside) and Lotaustiallin (methyl-Linamarin). Linamarin is the most abundant cyanoglucosides present in cassava cells (Conn, 1973) and may generate the equivalent to 0.2-100 mg of HCN per 100 g of fresh cassava following cellular lyses (Bradbury et al., 1991). The cassava effluent has been found to increase the number of organisms in the soil ecosystem which may be associated with increase in the soil pH, organic carbon and total nitrogen (Ogboghodo et al., 2001).

WASTE MANAGEMENT IN CASSAVA STARCH FACTORIESWaste from cassava processing may be solid or liquid. The brown peel of cassava roots, known as periderm, varies between 2-5% of the root total. The solid waste is made up of fibrous root materials and contains starch that physically could not be extracted. The process of starch extraction from cassava requires large quantity of water resulting in the release of a significant quantity of effluents (Balagopalan and Rajalakshmy, 1998). It is common for factories to discharge the effluents into the nearby rivers, drainage channels, crop fields or to the land adjacent the factories. These effluents pose a serious threat to the environment and quality of life in rural areas.Wide variations were observed in physical and chemical constituents of primary and secondary effluents from cassava starch factories. (Manilal et al., 1991) observed that the chemical oxidation demand (COD) ranged between 33,600 & 38,223mgl-1 in the primary effluents, whereas in the secondary effluents, the range was only 3800-9050mgl-1.The biological oxidation demand (BOD) was in the range of 13,200-14,300mgl-1 in the primary effluents. The corresponding figures for the secondary effluents were 3,600-7,050mgl-1. The acidity of the effluent ranged between pH 4.5 & 4.7. Nitrogen and phosphorus are the main nutrients contributing to the stability of organic waste and the analysis revealed low nitrogen content indicating necessity for the enrichment of the effluent to reduce the BOD and COD (Manilal et al., 1991).Balagopalan and Rajalakshmy, 1998 observed that the concentration of total cyanoglucosides in the effluents ranged between 12.9mgl-1 & 16.6mgl-1 in the case of initial samples, whereas in the case of final waste samples, the concentration ranged between 10.4mgl-1 & 27.4mgl-1. A high concentration of cyanide was observed in the ground water source near the processing factories ranging between 1.2mgl-1 & 1.6mgl-1. Initial settling, anaerobiosis, filtration through sand & charcoal and aeration can reduce the pollution load to the desired level (Balagopalan et al., 1994)

Microorganisms can grow on substrates containing cyanides by anaerobic metabolism, or by using an aerobic respiration chain as an alternative pathway (Cereda et al., 1991). In both pathways, HCN is eliminated from the substrate, and converted into a non-toxic product (Jensen et al., 1979). This enzymatic cyanide-removing property can be exploited for the detoxification of cyanide-rich cassava wastewater and industrial residues. These residues currently cause serious environmental problems in many cassava flour producing plants in Brazil, the largest producer worldwide, and in many African, Latin American and Asian countries (Romero et al., 2002), where cassava products are an important input for human diet.BACTERIABacteria are single-cell organisms and the most numerous denizens of agriculture, with populations ranging from 100million to 3billion in a gram. They are capable of very rapid reproduction by binary fission (dividing into two) in favorable conditions. One bacterium is capable of producing 16 million more in just 24 hours. Most soil bacteria live close to plant roots and are often referred to as rhizobacteria. Bacteria live in soil water, including the film of moisture surrounding soil particles, and some are able to swim by means of flagella. The majority of the beneficial soil-dwelling bacteria need oxygen (and are thus termed aerobic bacteria), whilst those that do not require air are referred to as anaerobic, and tend to cause putrefaction of dead organic matter. Aerobic bacteria are most active in a soil that is moist (but not saturated, as this will deprive aerobic bacteria of the air that they require), and neutral soil pH, and where there is plenty of food (carbohydrates and micronutrients from organic matter) available. Hostile conditions will not completely kill bacteria; rather, the bacteria will stop growing and get into a dormant stage, and those individuals with pro-adaptive mutations may compete better in the new conditions.FUNGIFungi are microscopic cells that usually grow as long threads or strands called hyphae, which push their way between soil particles, roots, and rocks. Hyphae are usually only several thousandths of an inch (a few micrometers) in diameter. Single hyphae can span in length from a few cells to many yards. Hyphae sometimes group into masses called mycelium or thick, cord-like rhizomorphs that look like roots. Fungi perform important services related to water dynamics, nutrient cycling, and disease suppression. Along with bacteria, fungi are important as decomposers in the soil food web. They convert hard-to-digest organic material into forms that other organisms can use. Fungal hyphae physically bind soil particles together, creating stable aggregates that help increase water infiltration and soil water holding capacity.Soil fungi can be grouped into three general functional groups based on how they get their energy. Decomposers saprophytic fungi convert dead organic material into fungal biomass, carbon dioxide (CO2), and small molecules, such as organic acids. These fungi generally use complex substrates, such as the cellulose and lignin, in wood, and are essential in decomposing the carbon ring structures in some pollutants. A few fungi are called sugar fungi because they use the same simple substrates as do many bacteria. Like bacteria, fungi are important for immobilizing, or retaining, nutrients in the soil. In addition, many of the secondary metabolites of fungi are organic acids, so they help increase the accumulation of humic-acid rich organic matter that is resistant to degradation and may stay in the soil for hundreds of years. Mutualists the mycorrhizal fungi colonize plant roots. In exchange for carbon from the plant, mycorrhizal fungi help solubolize phosphorus and bring soil nutrients (phosphorus, nitrogen, micronutrients, and perhaps water) to the plant. One major group of mycorrhizae, the ectomycorrhizae (see third photo below), grows on the surface layers of the roots and are commonly associated with trees. The second major group of mycorrhizae is the endomycorrhizae that grow within the root cells and are commonly associated with grasses, row crops, vegetables, and shrubs. Arbuscular mycorrhizal (AM) fungi are a type of endomycorrhizal fungi (see fourth photo below). Ericoid mycorrhizal fungi can by either ecto- or endomycorrhizal. The third group of fungi, pathogens or parasites, cause reduced production or death when they colonize roots and other organisms. Root-pathogenic fungi, such as Verticillium, Pythium, and Rhizoctonia, cause major economic losses in agriculture each year. Many fungi help control diseases. For example, nematode-trapping fungi that parasitize disease-causing nematodes, and fungi that feed on insects may be useful as biocontrol agents.

2.2ANTIBIOTICSThe control of microorganism is critical for the prevention and treatment of diseases. Modern medicine is dependent on chemotherapeutic agents, chemical agents that are used to treat infections. Most of these agents are antibiotics, microbial products or their derivative that can kill susceptible microorganism or inhibit their growth. Some bacteria and fungi are able to naturally produce many of the commonly employed antibiotics. In contrast, several important chemotherapeutic agents such as sulfonamides, trimethoprim, chloramphenicol, ciprofloxacin and dapsones are synthetic while increasing number of antibiotics are semi synthetic.Antibiotics vary in their effectiveness, many are narrow-spectrum drugsthat is they are effective only against a limited variety of pathogens. Others are broad-spectrum drugsthey are able to attack many different kinds of pathogens. Drugs may also be classified based on the general microbial group they act against: antibacterial, antifungal, antiprotozoan, and antiviral. Some antibiotics can be cidal or static in action. Static agents reversibly inhibit growth, if the agent is removed, the microorganism will recover and grow again. Although a cidal agent kills the target pathogen, its activity is concentration dependent and the agent may only be static at low levels.2.2.1CLASSIFICATION OF ANTIBIOTICSThere are many classes of antibiotics available to modern medicine today, classification may be based on route of administration, and mode of action (static or cidal) etc. most commonly used groups of antibiotics is the: Penicillins, Cephalosporins, Aminoglycosides, Macrolides, Quinolones and fluoroquinolones etc.PenicillinPenicillin is cidal in its mode of action, a narrow-spectrum antibiotic that functions to inhibit transpeptidization enzyme involved in cross-linking the polysaccharide chains of the bacterial cellwall peptidoglycan. Penicillin is used to treat skin infections, urinary tract infections; gonorrhea etc. examples include Penicillin G, V, methicillin.CephalosporinCephalosporins are a family of antibiotics originally isolated in 1948 from the fungus Cephalosporium. They contain a -lactan structure that is similar to that of penicillin. Cephalosporin is also cidal in action, it is a broad-spectrum antibiotic that functions to inhibit transpeptidization enzyme involved in cross-linking the polysaccharide chains of the bacterial cellwall peptidoglycan. Cephalosporin is used to treat pneumonia, strep throat, staphylococcus infection; various skin infection etc. examples include Cephalothin, Cefoxitin, Ceftriaxone.

AminoglycosidesThey are also cidal in action, a broad-spectrum antibiotic that acts by binding to small ribosomal subunits (30S) and interfere with protein synthesis by directly inhibiting synthesis and causing misreading of mRNA. Aminoglycosides are given for a short time periods and are injected intravenously rather than orally because they are easily broken down in the stomach. Examples include Neomycin, Gentamicin, and Streptomycin.MacrolidesThese antibiotics are derived from Streptomycin bacteria. They are bacteriostatic and a broad-spectrum antibiotic, binding to 23S rRNA of large ribosomal subunit (50S) to inhibit peptide chain elongation during protein synthesis. They are used to treat gastrointestinal upset, respiratory tract infection etc. examples include Erythromycin, Clindamycin.Erythromycin is a relatively broad-spectrum antibiotic effective against gram-positive bacteria, mycoplasmas and a few gram-negative bacteria. TrimethoprimTrimethoprim is a synthetic antibiotic that also interferes with the production of folic acid. It does so by binding to dihydrofolate reductase (DHFR), the enzyme responsible for converting dihydrofolic acid to tetrahydrofolic acid, competing against dihydrofolic acid substrate. It is a broad-spectrum antibiotic often used to treat respiratory and middle ear infections, urinary tract infections, and travelers diarrhea

2.3ANTIBIOTICS RESISTANCE BY MICROORGANISMSAntibiotics are very important to medicine but it is quite unfortunate that microorganisms have been able to adapt themselves to co-habiting with antibiotics and subsequently developing resistance to them (Walsh et al., 2004). When microorganisms are continually exposed to the same antibiotics, they find ways of adapting themselves to such antibiotic and this renders the drug ineffective against them. Transfer of resistance gene can be transferred by conjugation, transduction or transformation (Walsh, 2003).Widespread use of antibiotics both inside and outside medicine is playing a significant role in the emergence of resistant organism (Furaya and Lowy, 2006). Drugs frequently have been overused in the past. It has been estimated that over 50% of the antibiotic prescriptions in the hospital are given without clear evidence of infection or adequate medical indication (Payne et al., 2004). Many physicians have administered antibacterial drugs to patients with colds, influenza, viral pneumonia, and other viral diseases.A recent study showed that over 50% of patients diagnosed with colds and upper respiratory infections and 66% of those with chest colds (bronchitis) are given antibiotics, even though over 90% of those cases are caused by viruses (Furaya and Lowy, 2006).Frequently antibiotics are prescribed without culturing and identifying the pathogen or without determining bacterial sensitivity to the drug (Harbath et al., 2005). Toxic, broad-spectrum drugs are sometimes given in place of narrow-spectrum drugs as a substitute for culture and sensitivity testing, with the consequent risk of dangerous side effects, opportunistic infections, and the selection of drug-resistant mutants (Payne et al., 2005). The situation is made worse by patients not completing their course of medication. When antibiotic treatment is ended too early, drug-resistant mutants may survive. Antibiotics are used often in rearing animals for food and this use among others leads to creation of resistant strains. In supposedly well-regulated human medicine, the major problem of emergence of resistant strains is due to misuse and overuse of antibiotics by doctors as well as patients and it has been discovered that infections caused by resistant microorganism often fail to respond to standard treatment resulting in prolonged illness and greater risk of death (Walsh et al., 2004).

CHAPTER THREE3.0MATERIALS AND METHODS3.1MATERIALSVarious materials were involved in the analysis, these materials include: Petri dishes commonly used for the holding the agar medium on which the organisms are to be grown, Syringes and needles which are employed in dispensing accurate measures of liquids such as distilled water involved in the analysis, Measuring cylinder used to measure a precise amount of liquids, Conical flasks used for holding the prepared medium, Ethanol which is commonly used to swab the working environment and also to supply fuel to spirit lamps, Test-tubes for holding distilled water needed for serial dilution, Cotton wool, aluminium foil, paper tape whose functions ranges from swabbing and plugging of flasks mouth, wrapping of objects air-tightly to labeling, Wire loop for transferring of organism, Weighing balance used for taking accurate measurements of medium to be used. Agars used include: nutrient agar for culturing bacteria, PDA (potato dextrose agar) for culturing yeasts and moulds.

3.2COLLECTION OF SAMPLESSoil samples from three (3) different spots from Lautech gari processing industry were collected: The first point or location was the point of discharge of the cassava effluent or wastewater i.e. where the cassava wastewater drains into and this was labeled Sample A. Next location was one hundred meters (100m) away from the point of discharge of the cassava effluent or wastewater i.e. 100m along the part of flow of cassava wastewater and this was labeled as Sample B while the last location was soil sample from a neutral source that has not witnessed any form of cassava effluent discharge or contamination and this was labeled Sample C.Sample A which is the soil sample from the cassava effluent was collected using a sterilized spoon, it was collected into a sterile glass container, and the same procedure was used for Samples B and C but with different sterilized glass containers and spoons involved. The samples after collection were transported into the laboratory in a sterile black polythene bag for subsequent microbial analysis.3.3CULTURING OF MICROORGANISMSMedia were prepared according to manufacturers instructions printed on the container, and were sterilized in an autoclave at 1210C for 1hour. The collected soil samples were homogenized using distilled water. 9mls of sterile distilled water was dispensed into each test-tube and subsequent plugging with cotton wool and aluminium foil; these tubes were then taken to the autoclave for sterilization at 121oc for 1hour and after sterilization, cooling was done.Serial dilution was done to thin out microbial population so that numbers of colonies that will be formed would not overpopulate the plate unto which they will be grown.Serial dilution was carried out for each sample using sterile distilled water as the diluent and this was done under aseptic condition. The aseptic condition was achieved by the thorough swabbing of the working environment with ethanol and cotton wool, the work table was also swabbed clean and lit with spirit lamps to prevent contamination from atmosphere. Three dilution factors for each soil sample were picked and used as inoculum for the culturing of the organisms using pour plate method. Incubation was done at 370c for 24hours for bacteria and 48hours for fungi. Observation and recording was done after the completion of the incubation period.ISOLATION OF ORGANISMSFrom the mixed culture, distinct colonies were picked and streaked unto a freshly prepared media under aseptic condition. Subculturing was done repeatedly to obtain pure isolates which were stored on slants. IDENTIFICATION OF ISOLATESThe isolates were subjected to various biochemical tests for identification according to specification of (Starr et al., 1981) in the laboratory

Figure showing arrays of plates in a lamina flow chamber and an isolate growing on the plate

3.7.2BIOCHEMICAL TESTSThe following are the biochemical tests performed on the isolates;3.7.2.1CATALASE TESTA slide test was employed, a small quantity of the cultures were put on a glass slide, a drop or two of hydrogen peroxide is then added to the slide. The presence of bubbles represents a positive (+ve) test while a negative test is signaled by the absence of bubbles.

3.7.2.2HYDROGEN SULPHIDE TESTExamine each SIM tube for the presence of a black color (nothing needs to be added). A black color indicates the presence of hydrogen sulphide (H2S) which combines with the peptonized iron in the SIM medium. The result is FeS iron sulphide which causes a blackening of the medium and this represents a positive test; the absence of a black color is a negative test.Fig. showing hydrogen sulphide test

3.7.2.3INDOLE TESTUse a dropper to place 5drops of Kovacs reagent onto the top of the SIM agar in each tube. If the amino acid tryptophan has been broken down by the enzyme tryptophanase to form indole, the kovacs reagent will combine with the indole to form a red color at the top of the agar and this represents a positive test. No color change in the kovacs reagent represent is a negative test.

Fig. showing indole test (notice the red color on the top of the agar)

3.7.2.4METHYL RED TESTUsing a Pasteurs pipette, add 10drops of methyl red pH indicator to each tube, swirl the tube gently to mix the drops into the broth. Examine each tube for color change. Bacteria that produce many acids from the breakdown of dextrose (glucose) in the MR-VP medium cause the pH to drop to 4.2. At this pH, methyl red is red. A red color represents a positive test. Bacteria that produce fewer acids from the breakdown of glucose drop the pH to 6.0. At this pH methyl red is yellow and this represents a negative test.

3.7.2.5OXIDASE TESTDrop 1-2 drops of oxidase reagent onto colonies of broth culture, watch out for gradual color change from pink to light purple and then to dark purple within 10-30seconds. Such a color change indicates the presence of the respiratory enzyme cytochrome C oxidase and this represents a positive test. No color change is a negative test.

3.7.2.6OXIDATION-FERMENTATION (O-F) GLUCOSE TESTO-F glucose medium contains the sugar glucose and pH indicator bromthymol blue. This indicator is green at the initial pH of 6.8, but turns to yellow at a pH of 6.0. if glucose is utilized, acids are produced and the pH drops, causing the bromthymol blue to turn from green to yellow. If both tubes (with or without oil) turn yellow, the test organism is said to be a facultative anaerobe able to use glucose in the presence or absence of oxygen. If only the tube without oil turns yellow, the test organism is considered an aerobe able to use glucose only when oxygen is present. No color change in either tube indicates that the test organism is unable to utilize glucose.

3.8ANTIBIOTIC SENSITIVITY TESTINGColonies from the slants were picked and used to inoculate appropriate broth culture (Nutrient broth for bacteria and Potato dextrose broth for fungi) and then incubated for less than 18hours. Fresh media were prepared and left overnight for surface moisture to dry up. Picking of colonies from the broth cultures was done using sterile applicator stick and proper swabbing unto the surface of the prepared plates was done. This was left for 1hour after which antimicrobial discs were applied using a sterile forceps; the discs were pressed down firmly to prevent falling off of the discs from the plates during incubation.For fungi, three concentrations of antifungal stock solution were prepared and sterile perforated filter papers were dipped into each stock solution using a sterile forceps. Picking and application of the discs unto the plates was done using a sterile forceps. Incubation was done and sensitivities were observed at 24hours and 48hours for bacteria while fungi were incubated for 48hours.After incubation, the zones of inhibition formed were measured in two perpendicular, planes with the averages determined. After this the results was interpreted using standard tables to determine if the bacteria are Sensitive (S), Intermediate (I) or Resistant (R) to the antimicrobial drugs.

CHAPTER FOUR

4.0RESULTS AND DISCUSSION4.1ISOLATED ORGANISMSA total number of twenty-five strains were isolated from all the samples; some of them are (Bacillus cereus, Bacillus subtilis, Pseudomonas aeruginosa, Listeria monocytogenes, E.coli etc), while fungal strains include; (Aspergillus niger, Aspergillus flavus and Rhizopus sp etc)

PLATE COUNT RESULTTable 1 shows the result from the colony count of each sample from different media.MacConkey AgarSAMPLES10-110-210-4

ATNC6736

BTNC7039

CTNC8019

PDASAMPLES10-110-210-4

ATNC4917

BTNC5236

C1339342

Nutrient AgarSAMPLES10-110-310-5

ATNCTNC96

BTNC9485

CTNCTNC86

4.2IDENTIFICATION OF ORGANISMSTable 2 shows the identified organisms obtained in the samples (bacteria and fungi).S/NCodesIsolates

1NA A-1Pseudomonas aeruginosa

2NA A-2 1Bacillus cereus

3NA A-22Bacillus subtilis

4NA A-31Bacillus subtilis

5NA A-32Bacillus subtilis

6NA A-53Pseudomonas aeruginosa

7NA A-54Bacillus subtilis

8NA B-11Listeria monocytogenes

9NA B-12E. coli

10NA B-31Listeria monocytogenes

11NA B-32E. coli

12NA B-52E. coli

13NA B-53E. coli

14NA C-31E. coli

15NA C-42Bacillus cereus

16NA C-42Bacillus cereus

17NA C-54Bacillus subtilis

18NA C-54E. coli

A11Aspergillus niger

A12Aspergillus flavus

A21Aspergillus niger

B11Rhizopus sp

B12Aspergillus niger

C2Aspergillus niger

Mycotene Result in mm

Fungi50g/ml100g/ml200g/ml

A11R1820

A12131519

A21153035

Fungi50g/ml100g/ml200g/mlB11RRRB1212.52022

C214.51921

4.3ANTIBIOTIC SENSITIVITY TESTINGTable 3 shows the sensitivity result for the isolated organisms from samples A, B, and C at 24hours and 48hours respectively.

SENSITIVITY TESTING RESULT (24 HOURS) SAMPLE A (CASSAVA EFFLUENT)SAMPLESCAZCRXGENCPROFLAUGNITAMPERYCTRCXC

-ve A31RR21.024.521.518.018.511.5---------

-ve A21RR19.024.023.513.517.010.5---------

+ve A32RR15.5---20.0R------11.5RR

-ve A22RR25.021.013.015.024.511.0---------

-ve A1 RR17.020.518.013.016.512.0---------

+ve A53RR16.0---20.0R------11.5RR

-ve A54RR18.023.020.512.015.011.0---------

SAMPLE B (100m from Cassava effluent)SAMPLESCAZ CRXGENCPR OFLAUGNITAMPERYCTRCXC

-ve B11RR16.523.521.512.513.5R---------

+ve B12RR21.5---28.0R------16.0RR

+ve B53RR16.0---17.0R------15.5RR

+ve B31RR13.0---20.0R------RRR

-ve B3211.016.019.025.020.020.015.010.0---------

-ve B52RR18.516.016.012.014.07.5---------

SAMPLE C (Normal soil)SAMPLESCAZCRXGENCPROFLAUGNITAMPERYCTRCXC

-ve C4220.015.516.017.022.06.013.0R---------

+ve C31RR14.5---14.5R------9.0RR

-ve C4220.017.017.025.025.08.09.0R---------

+ve C54RR14.0---24.0R------10.0RR

-ve C54RR17.020.520.010.014.010.5---------

SENSITIVITY TESTING RESULT AT (48 HOURS) SAMPLE A (CASSAVA EFFLUENT)SAMPLESCAZCRXGENCPROFLAUGNITAMPERYCTRCXC

-ve A31RR23.024.521.518.019.012.0---------

-ve A21RR19.024.024.014.517.013.0---------

+ve A32RR17.5---20.0R------12.5RR

-ve A22RR25.023.018.515.024.517.0---------

-ve A1 RR19.021.019.013.518.512.0---------

+ve A53RR17.0---21.0R------12.0RR

-ve A54RR20.027.525.013.017.012.0---------

SAMPLE B (100m from Cassava effluent)SAMPLESCAZ CRXGENCPR OFLAUGNITAMPERYCTRCXC

-ve B11RR17.524.523.013.014.0R---------

+ve B12RR28.0---32.0R------20.5RR

+ve B53RR16.5---20.0R------18.0RR

+ve B31RR15.5---21.0R------RRR

-ve B3212.517.021.027.023.021.015.015.5---------

-ve B52RR18.516.016.012.014.09.0---------

SAMPLE C (Normal soil)SAMPLESCAZCRXGENCPROFLAUGNITAMPERYCTRCXC

-ve C4222.019.019.020.025.07.515.0R---------

+ve C31RR16.0---21.0R------11.0RR

-ve C4222.517.019.025.025.58.014.0R---------

+ve C54RR15.0---27.0R------11.0RR

-ve C54RR19.026.026.013.516.011.0---------

NegativePositiveCaz- Ceftazidine 30g Caz- Ceftazidine 30gCrx-Cefuroxime30gCrx-Cefuroxime30gGen-Gentamycin10gGen-Gentamycin10gCpr-Ciprofloxacin5gCtr-Ceftriaxone300gOfl-Ofloxacin5gCxc-Cloxacilin5gAug-Augumentin30gAug-Augumentin30gNit-Nitrofurantoin300gOfl-Ofloxacin5gAmp-Ampicillin10gEry-Erythromycin5g

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