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CHAPTER ONE

1.0 INTRODUCTION

Cassava (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).

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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

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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 1950’s it became

apparent that strains of bacteria were becoming resistant to specific antibiotics, it was

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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.

1.1 STATEMENT OF THE PROBLEM

Cassava 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

1.2 AIMS AND OBJECTIVES

To isolate, characterize and carry out antimicrobial susceptibility test on soil microorganisms

present within the cassava mill industry.

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CHAPTER TWO

2.1 LITERATURE REVIEW

Cassava (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.

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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

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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

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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 FACTORIES

Waste 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

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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.

BACTERIA

Bacteria 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

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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.

FUNGI

Fungi 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,

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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.

2.2 ANTIBIOTICS

The 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 drugs—that is they are

effective only against a limited variety of pathogens. Others are broad-spectrum drugs—they are

able to attack many different kinds of pathogens.

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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.

2.2.1 CLASSIFICATION OF ANTIBIOTICS

There 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.

Penicillin

Penicillin 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.

Cephalosporin

Cephalosporins are a family of antibiotics originally isolated in 1948 from the fungus

Cephalosporium. They contain a β-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.

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Aminoglycosides

They 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.

Macrolides

These 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.

Trimethoprim

Trimethoprim 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

traveler’s diarrhea

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2.3 ANTIBIOTICS RESISTANCE BY MICROORGANISMS

Antibiotics 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

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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).

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CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1 MATERIALS

Various 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.

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3.2 COLLECTION OF SAMPLES

Soil 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.

3.3 CULTURING OF MICROORGANISMS

Media 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.

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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 ORGANISMS

From 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 ISOLATES

The isolates were subjected to various biochemical tests for identification according to

specification of (Starr et al., 1981) in the laboratory

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

3.7.2 BIOCHEMICAL TESTS

The following are the biochemical tests performed on the isolates;

3.7.2.1 CATALASE TEST

A 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.

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3.7.2.2 HYDROGEN SULPHIDE TEST

Examine 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

3.7.2.3 INDOLE TEST

Use a dropper to place 5drops of Kovac’s 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 kovac’s 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 kovac’s reagent represent is a

negative test.

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

3.7.2.4 METHYL RED TEST

Using a Pasteur’s 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.

3.7.2.5 OXIDASE TEST

Drop 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.

22

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.

3.7.2.6 OXIDATION-FERMENTATION (O-F) GLUCOSE TEST

O-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.

3.8 ANTIBIOTIC SENSITIVITY TESTING

Colonies 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

23

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.

24

CHAPTER FOUR

4.0 RESULTS AND DISCUSSION

4.1 ISOLATED ORGANISMS

A 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 RESULT

Table 1 shows the result from the colony count of each sample from different media.

MacConkey Agar

SAMPLES 10-1 10-2 10-4

A TNC 67 36

B TNC 70 39

C TNC 80 19

PDA

SAMPLES 10-1 10-2 10-4

A TNC 49 17

B TNC 52 36

C 133 93 42

25

Nutrient Agar

4.2 IDENTIFICATION OF ORGANISMS

Table 2 shows the identified organisms obtained in the samples (bacteria and fungi).

S/N Codes Isolates

1 NA A-1 Pseudomonas aeruginosa

2 NA A-2 1 Bacillus cereus

3 NA A-22 Bacillus subtilis

4 NA A-31 Bacillus subtilis

5 NA A-32 Bacillus subtilis

6 NA A-53 Pseudomonas aeruginosa

7 NA A-54 Bacillus subtilis

8 NA B-11 Listeria monocytogenes

9 NA B-12 E. coli

10 NA B-31 Listeria monocytogenes

11 NA B-32 E. coli

26

SAMPLES 10-1 10-3 10-5

A TNC TNC 96

B TNC 94 85

C TNC TNC 86

12 NA B-52 E. coli

13 NA B-53 E. coli

14 NA C-31 E. coli

15 NA C-42 Bacillus cereus

16 NA C-42 Bacillus cereus

17 NA C-54 Bacillus subtilis

18 NA C-54 E. coli

A11 Aspergillus niger

A12 Aspergillus flavus

A21 Aspergillus niger

B11 Rhizopus sp

B12 Aspergillus niger

C2 Aspergillus niger

Mycotene Result in ‘mm’

Fungi 50µg/ml 100µg/ml 200µg/ml

A11 R 18 20

A12 13 15 19

A21 15 30 35

27

Fungi 50µg/ml 100µg/ml 200µg/ml

B11 R R R

B12 12.5 20 22

C2 14.5 19 21

4.3 ANTIBIOTIC SENSITIVITY TESTING

Table 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)

SAMPLES CAZ CRX GEN CPR OFL AUG NIT AMP ERY CTR CXC

-ve A31 R R 21.0 24.5 21.5 18.0 18.5 11.5 --- --- ---

-ve A21 R R 19.0 24.0 23.5 13.5 17.0 10.5 --- --- ---

+ve A32 R R 15.5 --- 20.0 R --- --- 11.5 R R

-ve A22 R R 25.0 21.0 13.0 15.0 24.5 11.0 --- --- ---

-ve A1 R R 17.0 20.5 18.0 13.0 16.5 12.0 --- --- ---

+ve A53 R R 16.0 --- 20.0 R --- --- 11.5 R R

-ve A54 R R 18.0 23.0 20.5 12.0 15.0 11.0 --- --- ---

28

SAMPLE B (100m from Cassava effluent)

SAMPLES CAZ CRX GEN CPR OFL AUG NIT AMP ERY CTR CXC

-ve B11 R R 16.5 23.5 21.5 12.5 13.5 R --- --- ---

+ve B12 R R 21.5 --- 28.0 R --- --- 16.0 R R

+ve B53 R R 16.0 --- 17.0 R --- --- 15.5 R R

+ve B31 R R 13.0 --- 20.0 R --- --- R R R

-ve B32 11.0 16.0 19.0 25.0 20.0 20.0 15.0 10.0 --- --- ---

-ve B52 R R 18.5 16.0 16.0 12.0 14.0 7.5 --- --- ---

SAMPLE C (Normal soil)

29

SAMPLE

S

CAZ CRX GEN CPR OFL AUG NIT AMP ERY CTR CXC

-ve C42 20.0 15.5 16.0 17.0 22.0 6.0 13.0 R --- --- ---

+ve C31 R R 14.5 --- 14.5 R --- --- 9.0 R R

-ve C42 20.0 17.0 17.0 25.0 25.0 8.0 9.0 R --- --- ---

+ve C54 R R 14.0 --- 24.0 R --- --- 10.0 R R

-ve C54 R R 17.0 20.5 20.0 10.0 14.0 10.5 --- --- ---

SENSITIVITY TESTING RESULT AT (48 HOURS)

SAMPLE A (CASSAVA EFFLUENT)

SAMPLE

S

CAZ CRX GEN CPR OFL AUG NIT AMP ER

Y

CTR CXC

-ve A31 R R 23.0 24.5 21.5 18.0 19.0 12.0 --- --- ---

-ve A21 R R 19.0 24.0 24.0 14.5 17.0 13.0 --- --- ---

+ve A32 R R 17.5 --- 20.0 R --- --- 12.5 R R

-ve A22 R R 25.0 23.0 18.5 15.0 24.5 17.0 --- --- ---

-ve A1 R R 19.0 21.0 19.0 13.5 18.5 12.0 --- --- ---

+ve A53 R R 17.0 --- 21.0 R --- --- 12.0 R R

-ve A54 R R 20.0 27.5 25.0 13.0 17.0 12.0 --- --- ---

30

SAMPLE B (100m from Cassava effluent)

SAMPLES CAZ CRX GEN CPR OFL AUG NIT AMP ERY CTR CXC

-ve B11 R R 17.5 24.5 23.0 13.0 14.0 R --- --- ---

+ve B12 R R 28.0 --- 32.0 R --- --- 20.5 R R

+ve B53 R R 16.5 --- 20.0 R --- --- 18.0 R R

+ve B31 R R 15.5 --- 21.0 R --- --- R R R

-ve B32 12.5 17.0 21.0 27.0 23.0 21.0 15.0 15.5 --- --- ---

-ve B52 R R 18.5 16.0 16.0 12.0 14.0 9.0 --- --- ---

SAMPLE C (Normal soil)

SAMPLE

S

CAZ CRX GEN CPR OFL AUG NIT AMP ERY CTR CXC

-ve C42 22.0 19.0 19.0 20.0 25.0 7.5 15.0 R --- --- ---

+ve C31 R R 16.0 --- 21.0 R --- --- 11.0 R R

-ve C42 22.5 17.0 19.0 25.0 25.5 8.0 14.0 R --- --- ---

+ve C54 R R 15.0 --- 27.0 R --- --- 11.0 R R

-ve C54 R R 19.0 26.0 26.0 13.5 16.0 11.0 --- --- ---

31

Negative Positive

Caz- Ceftazidine 30µg Caz- Ceftazidine 30µg

Crx- Cefuroxime 30µg Crx- Cefuroxime 30µg

Gen- Gentamycin 10µg Gen- Gentamycin 10µg

Cpr- Ciprofloxacin 5µg Ctr- Ceftriaxone 300µg

Ofl- Ofloxacin 5µg Cxc- Cloxacilin 5µg

Aug- Augumentin 30µg Aug- Augumentin 30µg

Nit- Nitrofurantoin 300µg Ofl- Ofloxacin 5µg

Amp- Ampicillin 10µg Ery- Erythromycin 5µg

32

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