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 7­13 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).

1

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 ton­1 of residues from washing

and 3.68 m3 ton­1 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.

2

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).

3

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 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 non­pathogenic that are present within the cassava mill

factories

4

1.2 AIMS AND OBJECTIVES

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

present within the cassava mill industry.

5

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 7­13 months

based on the cultivars planted (Cook, 1985; Taye, 1994). The tubers are quite rich in carbohydrates

(85­90%) 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

6

production of 35­40 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 (20­30%) 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 10­500 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 sub­lethal

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 iodine­deficient 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

7

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 sub­products, 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,

8

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 non­edible 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 (alpha­hydroxyisobutyronitrile­beta­D­glucopyranoside)

and Lotaustiallin (methyl­Linamarin). Linamarin is the most abundant cyanoglucosides present in cassava

cells (Conn, 1973) and may generate the equivalent to 0.2­100 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 2­5% 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

9

(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 & 38,223mgl­1 in the primary effluents, whereas in the

secondary effluents, the range was only 3800­9050mgl­1.

The biological oxidation demand (BOD) was in the range of 13,200­14,300mgl­1 in the primary

effluents. The corresponding figures for the secondary effluents were 3,600­7,050mgl­1. 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 12.9mgl­1 & 16.6mgl­1 in the case of initial samples, whereas in the case of

final waste samples, the concentration ranged between 10.4mgl­1 & 27.4mgl­1. A high concentration of

cyanide was observed in the ground water source near the processing factories ranging between

1.2mgl­1 & 1.6mgl­1. Initial settling, anaerobiosis, filtration through sand & charcoal and aeration can

reduce the pollution load to the desired level (Balagopalan et al., 1994)

10

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 non­toxic product (Jensen et al., 1979).

This enzymatic cyanide­removing property can be exploited for the detoxification of cyanide­rich

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 single­cell 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 soil­dwelling 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

11

kill bacteria; rather, the bacteria will stop growing and get into a dormant stage, and those individuals

with pro­adaptive 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, cord­like “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

hard­to­digest 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

12

fungi are organic acids, so they help increase the accumulation of humic­acid 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. Root­pathogenic fungi, such as Verticillium, Pythium,

and Rhizoctonia, cause major economic losses in agriculture each year. Many fungi help control

diseases. For example, nematode­trapping fungi that parasitize disease­causing 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.

13

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 narrow­spectrum drugs—that is they are

effective only against a limited variety of pathogens. Others are broad­spectrum drugs—they 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.

2.2.1 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

14

Penicillin is cidal in its mode of action, a narrow­spectrum antibiotic that functions to inhibit

transpeptidization enzyme involved in cross­linking 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 β­lactan structure that is similar to that of penicillin.

Cephalosporin is also cidal in action, it is a broad­spectrum antibiotic that functions to inhibit

transpeptidization enzyme involved in cross­linking 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.

Aminoglycosides

They are also cidal in action, a broad­spectrum 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

15

These antibiotics are derived from Streptomycin bacteria. They are bacteriostatic and a

broad­spectrum 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 broad­spectrum antibiotic effective against gram­positive bacteria,

mycoplasmas and a few gram­negative 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 broad­spectrum antibiotic

often used to treat respiratory and middle ear infections, urinary tract infections, and traveler’s diarrhea

16

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 co­habiting 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, broad­spectrum drugs are

sometimes given in place of narrow­spectrum drugs as a substitute for culture and sensitivity testing, with

the consequent risk of dangerous side effects, opportunistic infections, and the selection of

drug­resistant mutants (Payne et al., 2005). The situation is made worse by patients not completing

17

their course of medication. When antibiotic treatment is ended too early, drug­resistant 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 well­regulated 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).

18

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,

Test­tubes 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 air­tightly to labeling,

Wire loop for transferring of organism,

Weighing balance used for taking accurate measurements of medium to be used.

19

Agars used include: nutrient agar for culturing bacteria, PDA (potato dextrose agar) for

culturing yeasts and moulds.

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 test­tube and subsequent plugging

20

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 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

21

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.

22

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

23

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.

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 MR­VP 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.

24

3.7.2.5 OXIDASE TEST

Drop 1­2 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 10­30seconds. 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 OXIDATION­FERMENTATION (O­F) GLUCOSE TEST

O­F 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

25

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.

26

CHAPTER FOUR

4.0 RESULTS AND DISCUSSION

4.1 ISOLATED ORGANISMS

A total number of twenty­five 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

SAMPLE

S

10­1 10­2 10­4

A TNC 67 36

B TNC 70 39

C TNC 80 19

27

PDA

SAMPLES 10­1 10­2 10­4

A TNC 49 17

B TNC 52 36

C 133 93 42

Nutrient Agar

SAMPLES 10­1 10­3 10­5

A TNC TNC 96

B TNC 94 85

C TNC TNC 86

4.2 IDENTIFICATION OF ORGANISMS

28

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

S/

N

Codes Isolates

1 NA A­1 Pseudomonas aeruginosa

2 NA A­2 1 Bacillus cereus

3 NA A­22 Bacillus subtilis

4 NA A­31 Bacillus subtilis

5 NA A­32 Bacillus subtilis

6 NA A­53 Pseudomonas aeruginosa

7 NA A­54 Bacillus subtilis

8 NA B­11 Listeria monocytogenes

9 NA B­12 E. coli

10 NA B­31 Listeria monocytogenes

11 NA B­32 E. coli

12 NA B­52 E. coli

13 NA B­53 E. coli

14 NA C­31 E. coli

15 NA C­42 Bacillus cereus

16 NA C­42 Bacillus cereus

17 NA C­54 Bacillus subtilis

18 NA C­54 E. coli

29

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

Fungi 50µg/ml 100µg/ml 200µg/ml

B11 R R R

B12 12.5 20 22

30

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)

SAMPLE

S

CAZ CRX GEN CPR OFL AUG NIT AMP ER

Y

CTR CX

C

­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 ­­­ ­­­ ­­­

31

SAMPLE B (100m from Cassava effluent)

SAMPLES CAZ CRX GEN CPR OFL AUG NIT AMP ERY CT

R

CX

C

­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)

32

SAMPLE

S

CAZ CRX GEN CPR OFL AUG NIT AMP ERY CT

R

CX

C

­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 CX

C

­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

33

­ve A54 R R 20.0 27.5 25.0 13.0 17.0 12.0 ­­­ ­­­ ­­­

SAMPLE B (100m from Cassava effluent)

SAMPLES CAZ CRX GEN CPR OFL AUG NIT AMP ERY CT

R

CX

C

­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 CT

R

CX

C

­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 ­­­ ­­­ ­­­

34

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

35

REFERENCES

Adewoye, S.O; Fawole O.O.; Owolabi, O.D.; and Omotosho, J.S. (2005). Toxicity of

Cassava Wastewater Effluents to African Catfish, Clarias gariepinus­Burchell, 1822.E thiop.

J.Sc 28 (2): Pg. 180­194.

Akani, N.P.; Nmelo, S.A.; and ihemanandu, I.N. (2006). Effects of cassava processing

effluents on the microbial population and physiochemical properties of loamy soil in Nigeria. 10th

Annal. Conf. Nig Soc.

Anderson, J. M., and Inesor, P. (1983). Interaction between soil arthropods and

microorganisms in carbon, nitrogen and mineral element fluxes from decomposing leaf litter in: J.

A. Lee, McNeils and Rorison, I. H. (eds), Nitrogen as an Ecological Factor Pg.413­432.

Blackwell Scientific Publications, Oxford.

Antai, S.P., and Mbongo, P.M. (1994). Utilization of cassava peels as substrate for crude

protein formation. Plant foods human Nutr. 46: Pg. 345­451.

Akintonwa, A.; Tunwashe, O.; and Onifade, A. (1994). Fatal and non­fatal acute poisoning

attributed to cassava­ based meal. Acta Horticulturae. 375: Pg. 323­329.

36

Balagopalan,C., and Rajalakshmy, L. (1998). Cyanogen accumulation in the environment during

processing of cassava (Manihot esculenta Crantz) for starch & sago. Water, air and solid

pollution. 102. Pg. 407­413.

Bencini, M. C.(1991). Post – harvest and processing technologies of African staple food. A Technical Compendium. FAO Agricultural Service Bulletin 89. Rome: FAO.

Bradbury, J.H.; Egan, S.V.; and Lynch, M.J. (1991). Analysis of cyanide in cassava using acid

hydrolysis of cyanogenic glucosides. J. Sci. Food Agric.55. Pg. 277­290.

Bradbury, J. H. 2006. Simple wetting method to reduce cyanogens content of cassava flour. Journal of Food Composition Analysis. 19: Pg. 388­393.

Bokanga, M.; Ekanayake, I. J.; Dixon, A. G. O.; and Porto, M. C. M. (1994).

Genotype­environment interactions for cyanogenic potential in cassava. Acta Horticulturae.

375: Pg. 131­139.

Cereda, M.P. (2001). Caracterização dos subprodutos da industrialização da mandioca. In:

Cereda, M.P. (Ed.). Manejo, uso e tratamento de subprodutos da industrialização da

mandioca. Fundação Cargill, São Paulo, Pg.13­37.

Cereda, M.P.; Brasil, O.G.; Fioretto, A.M.C. (1981). Microrganismos com respiração

resistente ao cianeto isolados de líquido residual de fecularia. YTON 41. Pg. 197­201.

Conn, E.E. (1973). Biosynthesis of cyanogenic glycosides. Biochem.

Soc. Symp. 38: Pg. 277­302.

37

Cook, K. (1985). A New potential for neglected crops. Westview Press, Boader Co. USA

Conn, E.E. (1994). Cyanogenesis ­ a personal perspective. Acta Hortic. 375: Pg. 31­41.

Cowan, S.T., and Steel, K.J. ( 1990). Manual for the identification of Medical Bacteria.

Cambridge University press.

Cumbana, A.E., Mirione, J. C. and Bradbury, J.H.(2007). Reduction of cyanide content of

cassava flour in Mozambique by wetting method. Food Chemistry 101: 894­897l.

Daramola, B.,and Osanyinlusi, S.A. (2006). Investigation on modification of cassava starch

using active components of ginger roots. Afr. J. Biotechnology. 4: pg. 1117­1123.

Dindal, D. L.(1978). Soil organisms and stabilizing waste. J. Waste Recycling:19: Pg.8–11.

Eggleston, G.; Bokanga, M.; and Jean, Y. W. (1992). Traditional African methods for cassava

processing and utilisation and research needs. In M. O. Akoroda and O. B. Arene (Eds.),

Proceedings 4th Triennial Symposium, International Society for Tropical Root

Crops—Africa Branch, (pp. 3–6). Kinshasa, Zaire. December 5–8, 1989.

Furaya, E.Y., and Lowy, F.D. (2006). Antimicrobial­resistant bacteria in the community setting.

Nature Rev. Microbiol. 4: pg. 36­45.

Harbath, S., and Samore, M.H. (2005). Antimicrobial resistance determinants and future

control. Emerg. Infect. Dis. 11: Pg. 794­801.

38

Horsfall, M. Jr.; Abia, A.A.; and Spiff, A. I. (2006). Kinetic studies on the adsorption of

Cd2+, Cu2+ and Zn2+ ions from aqueous solutions by cassava (Manihot esculanta Crantz)

tuber bark waste. Bioresearches Technology 97: 283­291.

Howlett, W. P., and Konzo (1994). A new human disease entity. Acta Horticulturae. 375.

Pg. 323­329.

IITA, 2005. The uses of Cassava. Published by the Integrated Cassava Project of the

International Institute of Tropical Agriculture.

Isabirye, M., G.; Ruysschaert, L.; Van­ Linden, J.; Maguada M. K.; and Deckers J. (2007).

Soil losses due to cassava and sweet potatoes harvesting: a case study from low input traditional

agriculture. Soil tillage resources. 92: 96­103.

Jensen, H.L., and Abdel­Ghafar, A.S. (1979). Cyanuric acid as nitrogen sources for

microorganisms. Arch. Microbiol. 67: Pg. 1­5.

Jones, D.A. (1998). Why are so many plants cyanogenic? Phytochem.47, Pg. 155–162.

Jyothi, A. N., Sasikiran, B.N and Balagopalan, C. (2005). Optimization of glutamic acid

production from cassava starch factory residues using Brevibacterium divaricatum. Process

Biochemistry 40: 3576­3579.

Kehinde, A. T. (2006). Utilization Potentials of Cassava in Nigeria: The Domestic and

industrial products. Food Reviews International 22:29–42

39

Klugman, K.P., and Lonks, J.R. (2005). Hidden epidermic of macrolide­resistant

pneumococci. Emerg. Infect. Dis. 11: Pg. 802­807.

Lynam, J. R. (1993). Potential impact of biotechnology on cassava production in the 3rd

World: In. Hillock R. J., Thresh M. J. and Bellotti, A. C. Cassava: Biology; Production and

Utilization CABI International Oxford: 22 – 30.

Muro, M.A., and Luchi, M.R. (1989). Preservação de microrganismos. Fundação Tropical

de Pesquisas e Tecnologia “André Tosello”, Campinas.

Niessen, T. V. (1970). Biological degradation of hydrocarbons with special references to soil

contamination. Pl. Arl. 74: Pg. 391–405.

Onabowale, S.O. (1988). Processing of cassava for poultry feeds. In Proceedings of a

National Workshop on Alternative Livestock Feed Formulations in Nigeria, November,

21–25, Ilorin, Nigeria. Ed. Babatunde, G.M. Pg. 460–472.

Oboh, G.; Akindahunsi, A.A.; and Oshodi, A.A. (2002). Nutrient and anti­nutrient content of

Aspergillus niger fermented cassava products (flour and gari). J. Food compo. Anal. 15: Pg.

617­622.

Oboh, G., and Akindahunsi A.A. ( 2003a). Biochemical changes in cassava products subjected

to Saccaromyces cerevisae solid media fermentation. Food Chem. 82:599­602.

40

Oboh, G., and Akindahunsi A.A. ( 2003b). Chemical changes in cassava peels fermented with

mixed culture of Aspergillus niger and two species of Lactobacillus integrated Bio­system.

Applied Trop. Agric. 8: Pg. 63­68.

Oyewole, O.B., and Odunfa, S.A. (1992). Characterization and distribution of lactic bacteria in

cassava fermentation during fufu production. J.Appl. Bacteria. 68:148­152.

Oboh, G. ( 2005). Isolation and characterization of amylase from fermented cassava

wastewater. Afr. J. Biotechnol. 4: Pg.1117­1123.

Ogboghodo, I. A.; Osemwota, I. O.; Eke, S. O.; and Iribhogbe, A. E. (2001). Effect of

cassava (Manihot esculenta crantz) mill grating effluent on the textural, chemical and biological

properties of surrounding soils. World J. Biotechnol. 2(2), pg. 292–301.

Okafor, N. (1998). An integrated Bio­system for the disposal of cassava wastes. Integrated

Bio­system in zero Emission applications proceedings of the internet conference on internet

Bio­systems.

Osuntokun, B. O. (1994). Chronic cyanide intoxication of dietary origin and a degenerative

neuropathy in Nigerians. Acta Horticulturae. 375. Pg. 311­321.

Palmisano, M.M.; Nakamura, L.K.; Duncan, K.E.; Istock, C.A.; and Cohan, F.M. (2001).

Bacillus sonrensis sp. nov. A close relative of Bacillus licheniformis, isolated from soil in the

Sonoran Desert, Arizona. Int. J. Syst. Evol. Microbiol. 51: Pg. 1671­1679.

41

Payne, D., and Tomasz, A. (2004). The challenge of antibiotic­resistant bacterial pathogens:

The medical need, the market and prospects for new antimicrobial agents. Curr. Opin.

Microbiol. 7: Pg. 435­438.

Romero, M.C.; Hammer, E.; Cazau, M.C.; and Arambarri, A.M. (2002). Isolation and

characterization of biarylic structure – degrading yeasts: Hydroxylation potential of

dibenzofuran. Environ. Poll. 118: Pg. 379­382.

Raimbault, M. (1998). General and microbiology aspect of solid substrate fermentation. Elect.

J. Biotechnol.

Swift, M. J., Heal, O. W. and Anderson, J. M. (1979). Decomposition in Terrestrial

Ecosystem. Studies in Ecology Vol. 5. Blackwell Scientific Publications, Oxford, pp. 372.

Tian, G. L., Brussard and Kang, B. T. (1995). Breakdown of plant residues with contrasting

chemical composition under humid tropical conditions: Effects of earthworms and millipedes.

Soil Biol. Biochem. 27, Pg. 277–280.

Tweyongyere, R., and Katongole, I. ( 2002). Cyanogenic potential of cassava peels and their

detoxification for utilization as livestock feeds. Vet. Human Toxicol. 44: Pg.366­369.

Walsh, C. (2003). Where will the new antibiotics come from? Nature Rev. Microbiol. 1: Pg.

65­79.

Walsh, F.M., and Amyes, S.G.B. (2004). Microbiology and drug resistance mechanisms of

fully resistant pathogens. Curr. Opin. Microbiol. 7: Pg. 439­444.

42

43