ISOLATION AND IDENTIFICATION OF MICRORGANISMS

ASSOCIATED WITH DUMPSITES IN KEFFI MUNICIPAL.

BY

OYETUNJI EMMANUEL

NSU/NAS/PGD/MIB/021/15/16

DISSERTATION SUBMITTED TO THE SCHOOL OF

POSTGRADUATE STUDIES, NASARAWA STATE UNIVERSITY
KEFFI, IN PARTIAL FULFILLMENT OF THE REQUIREMENT FOR
THE AWARD OF NASARAWA STATE UNIVERSITY, KEFFI.

FACULTY OF NATURAL AND APPLIED DEPARTMENT OF

MICROBIOLOGY

SUPERVISOR:

ASSOCIATE PROF. M.D MAKUT

DECEMBER 2017
 DECLARATION

I, Oyetunji Emmanuel hereby declare that the Project Report entitled “Studies on Isolation
and Identification of Microorganisms Associated with Dumpsites in Keffi Municipal of
Nasarawa State” is done by me under the guidance of Associate Professor M.D Makut at
Nasarawa State University Keffi, and submitted in partial fulfilment of the requirements for
the award of Postgraduate Diploma in Microbiology and this work has not been submitted
elsewhere for any other degree.

------------------------------------- ------------------
Oyetunji Emmanuel Date
.

ii
 CERTIFICATION

This project titled isolation and identification of microorganisms associated with dumpsites in
keffi municipal, Nasarawa state of Nigeria, meets the regulations governing the area of
postgraduate diploma(PGD) in microbiology, faculty of natural and applied sciences,
department of microbiology, Nasarawa state university, keffi.

__________________ ________________
Associate Prof. M.D Makut Date
(Supervisor)

_______________________ ________________
Dr David Ishaleku Date
(Head of Department)

______________________ ________________
(External Examiner) Date

iii
 DEDICATION

This work is dedicated to God the Father, the Son and the Holy Spirit, my reason for living.

iv
 ACKNOWLEDGEMENTS

This project is a culmination of task undertaken by me during the course at Nasarawa state
university Keffi. Acknowledgement is not a mere formality or ritual but a genuine
opportunity to express the indebtedness to all those without whose active support and
encouragement this project wouldn’t have been possible.
First and foremost, my sincere appreciation goes to the Almighty God who spared my life.
I am highly indebted to Associate Prof. M.D Makut (Postgraduate Coordinator of
Microbiology Department), who was also my project supervisor for his continuous efforts in
developing a professional attitude within ourselves during the academic period.
My appreciation goes to Dr Ishaleku David (Head of Department Microbiology, Nasarawa
State University, Keffi) for his kind humane gesture and sacrifice rendered at various
periods in this study undergo this course. Sir I am indeed grateful.
Also my profound acknowledgement to all other lecturers of the department of
Microbiology, Faculty of Natural and Applied Sciences, Nasarawa State University, Keffi. I
also appreciate all the technical staff of this great department.
I am grateful to Mr. Istifanus Nkene whose valuable guidance and support constant
encouragement at every stage of work.
My course mates, just to mention a few: Balogun George, Ayobami Joshua, Olofinlade
Margret, Bunmi Ogunbiyi, Odunayo and my honourable class representative.
Diction is not enough to express my gratitude to my Spouse who has shown her
unconditional support and enormous sacrifice throughout this period. Indeed you are a rare
gem.

v
 ABSTRACT

A total of 15 soil samples were collected from five different waste-dumpsites. Standard
methods were used to isolate and identify viable bacteria and fungi from the samples. The
bacteria isolated from the waste dump soil were Bacillus sp, Microccus sp, Pseudomonas
aeruginosa, Pseudomonas fluorescens and Serratia sp. Only Bacillus sp and Pseudomonas
aeroginosa were isolated from all the stations. The fungi isolated were Aspergillus niger,
Aspergillus flavus, Aspergillus fumigatus, Mucor sp, Pencillium sp and Trichoderma viridae.
Aspergillus niger, Aspergillus flavus and Mucor sp were the most common fungi with
percentage frequency of 100% degradation potential of these isolates is herby recommended
for further studies.

vi
 TABLE OF CONTENTS
Title Page i
Declaration ii
Certification iii
Dedication iv
Table of Contents v
CHAPTER ONE
INTRODUCTION

1.1 Background of the study 1

1.2 Statement of the problem 3
1.3 Justification of the Study 4
1.4 Aim and Objective 4
1.4.1 Aim of the Study 4
1.4.2 Objective 4
CHAPTER TWO
LITERATURE REVIEW

2.1 Municipal Solid Waste 5

2.2 Municipal Solid Waste Management 6
2.3 Characterization of Bacteria 8
2.4 Cultural Methods 8

CHAPTER THREE
MATERIALS AND METHODS

3.1 Study Area 9

3.2 Sample Collection 9
3.3 Materials and reagents 10
3.3.1 Isolation of Bacteria 10
3.3.2 Isolation of Fungi 10

vii
3.3.3 Identification of Bacteria 10
3.4 Gram Staining 12
3.5 Biochemical Tests for Bacterial Isolates 12
3.5.1 Catalase Test 12
3.5.2 Mannitol Fermentation 12
3.5.3 Methyl Red and Voges Proskauer’s Test MR VP Broth 13

3.5.4 Voges Proskauer Test 13

3.5.5 Urease Test 13
3.5.6 Citrate Test 13
3.5.7 Glucose Fermentation Test 14

CHAPTER FOUR
RESULTS

4.1 Isolation and identification of bacteria 15

4.2 Isolation and identification of fungi 15
4.3 Frequency of occurrence of bacteria 19
4.4 Frequency of occurrence of fungi 20

CHAPTER FIVE
DISCUSSION, CONCLUSION AND RECOMMENDATION

5.1 Discussion 24
5.2 Conclusion 24
5.3 Recommendation 25
Reference 26
Appendix 30

viii
 CHAPTER ONE
INTRODUCTION

1.1 Background of the Study

According to the Basal Convention of 1992, “Wastes” are substance or objects, which are

disposed of or are intended to be disposed of or are required to be disposed of by the

provisions of national law.”

In addition, waste can also be defined as material, substance, or by product eliminated or

discarded as no longer useful or required after the completion of a process. (Basal

Convention, 1992).

Solid waste means any garbage, refuse, sludge, from a waste water treatment plant, or air

pollution control facility and other discarded material including solid, liquid semi- solid, or

contained gaseous material resulting from industrial, commercial, mining and agricultural

operations, and from community activities, but does not includes dissolved materials in

domestic sewage or solid or dissolved material in irrigation return flow or industrial

discharges. In simple words solid waste are any discarded or abandoned materials.

Rapid population growth and urbanization in developing countries has led to people

generating enormous quantities of solid waste and consequent environmental degradation.

The waste is normally disposed in open dumps creating nuisance and environmental

degradation. Solid wastes cause a major risk to public health and the environment.

Management of solid wastes is important in order to minimize the adverse effects posed by

their indiscriminate disposal, (Rajbanshi, 2008).

Domestic wastes such as containing a variety of materials thrown out from homes.

Also, food waste, Clothes, Waste paper, Glass bottles, Polythene bags, Waste metals, etc.

Commercial wastes: It includes wastes coming out from shops, markets, hotels, offices,

1
institutions, etc. ( Bremner et al.1989) . These include but not limited to waste paper,

packaging materials, cans, bottle, polythene bags, etc. Construction wastes: It included wastes

of materials. Wood, Concrete, Debris, etc. Biomedical wastes: It includes mostly waste

organic, Anatomical wastes, Infectious wastes, etc.

1.2 Statement of the Problem

Keffi city does not have sanitary landfill. Improper disposal of untreated municipal solid

wastes is not only harmful to human health but also constitute to ecological environment

(Yaliang, 1996). A waste is said to be hazardous if it is infectious, meaning containing

microorganisms or their toxins which are known or suspected to cause diseases in animals or

human (Yakowitz, 1988). Waste disposal poses threat to both man animal and the soil. Lire

chemical [Link] must be dispensed in the environment through water and

wind. Poisonous plants, insects, animals and indigenous pathogens are biologic hazards that

may be encountered at the waste sites (Khupe, 1996).

The composition of municipal solid waste in Keffi is food waste, paper cardboard, faeces,

screening residual, plastic, broken bottles. Batteries, textiles, bones, glass, wood and leave

ferrous metals, feather and rubber, non-ferrous metals, concretes and ceramics and hazardous

waste.

Waste management in developing countries is usually equated with land disposal or discharge

into bodies of water. This method of waste management is unscientific, causes nuisance to

the public, and constitute pollution and health hazards.

When waste is dumped on land, soli microorganisms including fungi and bacteria, readily

colonize the waste carrying out the degradation and transformation of degradable organic

materials in the waste (Stainer et al. 1989). Microorganisms in waste dump use the waste

constituents as nutrients. Thus detoxifying the material as their digestive process breakdown

complex organic molecules into simpler less toxic molecules. These metabolic activities can

2
be attributed to their high growth rate metabolism and their collective ability to degrade a

vast variety of naturally occurring organic materials. (Stainer et al., 1989).

1.3 Justification of the Study

The research work is very important to an individual and the entire Keffi metropolis on the

importance of waste disposal and the awareness sanitation- personal hygiene and

environmental management.

1.4 Aim and objectives

1.4.1 Aim of the study

The aim of this study is to isolate and identify microorganisms associated with degradation of

solid waste at dumpsites in Keffi metropolis. These organisms cam serve as potential bio

degraders of solid wastes.

1.4.2 Objectives of the study

The specific objectives of this study were as outlined below:

(a) To isolate and identify bacteria species associated with solid waste at dumpsites in

Keffi

(b) To isolate and identify fungi species associated with solid waste at dumpsites in Keffi

3
 CHAPTER TWO

LITERATURE REVIEW

2.0 Overview of Domestic Waste

The disposal of domestic, commercial and industrial garbage in the world is a problem that

continues to grow with human civilization and no method so far is completely safe

(Pacarynuk, et al, 2004).

Experience has shown that all forms of waste disposal have negative consequences on the

environment, public health, and local economies. Solid wastes are sources of environmental

pollution through introduction of chemical substances above their threshold limit into the

environment (Haq, et al,1999). Dumpsite is an old traditional method of waste disposal

similar to landfill method of waste management. Dumpsites are often established in disused

quarries, mining or excavated pits away from residential areas. Designated government

agency, corporate bodies and some individuals collect wastes routinely into these dumpsites

(Pacarynuk, et al. 2004).

2.1 Municipal Solid Waste

MSW is the waste generated in a community by household, commercial and/or institutional

activities with the exception of industrial and agricultural wastes hence, MSW includes

residential, institutional and commercial wastes which are considered to be non-hazardous

(Allison, 1977)

They also include food wastes, paper, cardboard, plastics, textiles, glass, metals, wood, street

sweepings, landscape and tree trimmings as well as general wastes from parks, beaches, and

other recreational areas. These wastes nevertheless often get contaminated with some

hazardous materials like the batteries and electronics. Local and regional factors, such as

4
climate and level of commercial activity, contribute to the variations in MSW composition.

(Berkeley, 1972). The content of organic matter in MSW in non-developed countries is found

to be higher due to the use of fresh and unprocessed vegetables. These are biodegradable and,

hence, have the potential to be converted into organic fertilizer for farm use. The

biodegradable fraction (paper, garden and food wastes) accounts for 53% of waste

composition (Allison, 1977).

The biodegradation of these wastes therefore, forms an important component of an integrated

solid waste management strategy, which will reduce both the harmful effects and volume of

the MSW requiring final disposal in a landfill. It is argued that city farming, through Urban

Agriculture (UA) programs, consumes the generated urban solid waste and reduces the

volume of waste to be collected and transported to distant dumps.

2.2 Municipal Solid Waste Management

Municipal Solid Waste Management (MSWM) in developing countries like Kenya faces lots

of challenges due to weak economies, inabilities to enforce environmental legislation, and

poor administrative capacities In most developing countries, the MSWM is of serious

concern especially due to rapid urbanization (Yakowtz, 1988).

If not properly, undertaken, inadequate management of wastes can cause harm to human

health and the environment.

Per capita daily municipal waste generation ranges from 2.75 to 4.0 Kg in high income

countries and 0.5 to 0.8 Kg in countries with low incomes (Khupe, 1996). Nairobi generates

about 4,000 tonnes of solid waste on daily basis (Khupe, 1996; Yaliang, 1996). While in

Kisumu, about 400 tonnes is generated every day. Only half of the estimated waste volume

generated daily in Nairobi is collected. With no means of safe disposal in the city, merely 850

tonnes of its daily waste reaches its main dump site, Dandora, with the other 2/3 of the total

5
waste generated hard to account for. Some of it finds its way into unofficial dumps, which

includes the Uthiru dumpsite. In Kisumu, only about 20 % of the 400 tonnes of solid waste

generated is collected and transported to the dumpsite.

Dandora dumping site covering an area of about 26 hectares has been documented to

negatively affect the health of thousands of Nairobi residents. Waste management is viewed

as one of Nairobi's key environmental concerns. A large percentage of solid waste in 8

Nairobi is managed by the private sector and NGOs through public-private partnerships, with

more than 2000 people earning their living through their engagement in waste resource

recovery.

The combination of urban organic wastes and UA is of particular interest in the modern urban

settings (Khupe, 1996).

On one hand, the need to reduce urban waste mesh well with the promotion of UA, since

urban and peri-urban farmers are in need of organic matter for soil improvement and animal

feed. On the other hand, cities and towns wish to conserve disposal space and reduce the

costs of MSWM. Besides composting, other MSWM practices include landfills, combustion/

incineration, source reduction, and recycling (Khupe, 1996).

Composting is preferable because it is cheap, prevents emissions of greenhouse gases,

reduces pollutants, saves energy, and conserves resources. Additionally, it can supply

valuable raw materials for agriculture, create jobs and reduce the need for new landfills and

combustors. Currently there are efforts to reduce waste streams to the major dumpsites in

cities through organized community and home composting in several countries.

2.3 Characterization of Bacteria

Bacteria can be described and classified in three major ways, namely; microscopic

examination, cultural characteristics, and molecular phylogenetics (Khupe, 1996).

6
The study of microbial diversity represents a major opportunity for advances in biology and

biotechnology. There is a massive capacity for genetic diversity of bacteria in the biosphere.

One of the major problems that hamper studying the bacterial diversity in the environment is

the inability to obtain many of bacteria in culture. Over the years, 17 diverse natural

microorganisms have yielded important biological materials useful to humans in medical,

industrial and agricultural fields. Recent progress in molecular microbial ecology shows that

the extent of microbial diversity in nature is far greater than previously thought.

2.4 Cultural Method

Pure culture techniques have been the core of standard microbiology (Allison, 1977).

Nonetheless, the traditional culture-dependent methods are strongly biased and limiting, as

only less than five per cent of bacteria can be cultured with the rest of them being

unamenable to this approach. Janssen and group revisited the question of culturability of soil

bacteria using simple methodologies, and have found that there is considerable scope for

improving both the apparent culturability and the phylogenetic range of bacteria that can be

cultivated (Allison, 1977).

Parallel study of widespread but previously uncultivated groups of organisms strongly

complement molecular ecological investigations and enhance research into the roles of

environmental bacteria and their biotechnological potentials. Though limiting in terms of the

number of cultivable bacteria, the culturing techniques still provide non-overlapping

information as compared to molecular screening.

7
 CHAPTER THREE

MATERIAL AND METHODS

3.1 Study Area

Keffi is a Local Government Area in Nasarawa State, Nigeria. It is located at North central of

Nigeria between the latitude 8º 90`N to 9º 00`N and longitude 7º 75`E to 8º 00`E. It has an

area of 138 km² and a population of 92,664 as at the 2006 census. Keffi lies within the guinea

savannah area of the North central Nigeria and the mean annual rainfall distribution in the

area ranges from 1000mm to 1200mm. Keffi metropolis is divided into ten wards which is to

have residential enclaves with about 4,000-5,000 household population. It is served with

common facilities like schools, shops, postal agency, hospitals/clinics, markets among others.

3.2 Sample Collection

Soil samples were collected from five different dumpsites in Keffi.(Angwan, Lambu, High

court, Emir’s Palace, School of Health and Dadin Kowa). The surface debris was removed

and the subsurface soil dug to a depth of about 15 cm with a hand shovel. Soil samples were

scooped from depths of 0-15 cm in sterile containers within the perimeter of each refuse

collection point (Isirimah et al., 2005). The samples were transported to the laboratory. The

samples were treated within 2 h of collection. The soil samples were sieved through a 0.2 mm

wire mesh, which was previously swabbed with 95% ethanol, to obtain fine soil particles

(USEPA, 1978).

3.3 Materials and Reagents

Nutrient Agar Medium, Ringer’s solution, test tubes, Petri plates, Measuring cylinders,

Beakers, Micropipettes, Conical flasks, Hand shovel, Sterile containers. 95% ethanol.

8
3.3.1 Isolation of bacteria.

1g of soil sample was measured in a conical flask, 9ml Ringer’s solution was added into it.

Serial dilution of stock solution was carried out. A 0.1ml amount was poured onto nutrient

agar plate. It was incubated at 370C for 24 hours as described by Brenner et al, (2005).

3.3.2 Isolation of fungi

1g of soil sample was measured in a conical flask, 9ml Ringers solution was added to

dissolve the solution. A 0.1ml was taken by micropipettes and pour plate on Sabouraud

dextrose agar, then it was incubated at 350C for 5 to 7 days. Observations of growth on agar

surface and on the reverse side of the agar plates were recorded.

3.3.3 Identification of bacteria

The biochemical test and identification of bacteria were carried out as described by

Cheesbrough (2001). In this method the citrate, oxidase, indole, catalase, urease, methyl red,

fructose, mannose and mannitol tests were carried out respectively, as described by Cowan

and Steel (2002). The isolates were later identified using Bergey’s manual described by

Brenner et al, (2005).

3.4.1. Gram staining

Thin smears of the isolated different colonies were prepared, air dried, and heat fixed. Smear

was covered with crystal violet for 60 seconds. The stain was washed off using distilled

water. The excess water was drained off. The smear was covered with Gram’s iodine

solution and kept for 60 seconds. The Gram’s iodine was poured off and the smear was

flooded with 95% alcohol for 30 seconds. The slide was washed with distilled water. The

counter stain Safranin was added to smear and was kept for 60 seconds. The stain was

washed gently for few seconds. The slide was air dried and, examined with a light

microscope under oil immersion.

9
3.5. Biochemical Tests of Bacterial Isolates

Biochemical tests were carried out according to standard methods. Brenner et al, (2005).

3.5.1 Catalase test

Two-three drops of 3% hydrogen peroxide were taken on a clean glass slide. One loop full of

the culture was just kept over the hydrogen peroxide. Slide was than observed for the

appearance or absence of gas bubbles. Occurrence of gas bubbles was recorded as positive

catalase test.

3.5.2 Sugar Utilization Test

The sugar utilization test for identification of bacteriaisolated from soil of dumpsite was

carried out as follows; 2-3 pure colonies of bacteria isolated were inoculated into 5ml of

medium containing different sugars such as glucose, lactose, fructose, mannose and

Mannitol. The tubes were incubated at 300C for 24 hours.

3.5.3. Methyl Red test and Voges-Proskauer’s test (MRVP Broth):

The culture was inoculated in the tubes containing MRVP broth, a control was also

maintained. The inoculated and control tubes were incubated at 370C, for 48 hours. After the

incubation was over, 12 drops of VP I reagent (alpha-napthalin) and 2-3 drops of VP II

reagent (KOH) was added to the inoculated and un-inoculated tubes. Tubes were shaken

gently for 30 seconds with the plugs off to expose the media to oxygen. The tubes were

observed for change in colour, the development of crimson to yellow colour is indicative of

VP positive test while no change in colour is a negative test.

3.5.4 Voges-Proskauer’s test:

The culture was inoculated in the tubes containing MRVP broth, a control was also

maintained. The inoculated and control tubes were incubated at 370C, for 48 hours. After the

incubation was over, 12 drops of VP I reagent (alpha-napthalin) and 2-3 drops of VP II

reagent (KOH) was added to the inoculated and un-inoculated tubes. Tubes were shaken

10
gently for 30 seconds with the plugs off to expose the media to oxygen. The tubes were

observed for change in colour, the development of crimson to yellow colour is indicative of

VP positive test while no change in colour is a negative test.

3.5.5 Urease test

The culture was inoculated in the tubes containing citrate test agar media, one control was

also maintained The inoculated tubes were incubated at 370C, for 24 hours. The tubes were

than observed for the change in colour of the media. A change in colour from green to blue

indicates a positive result, while no change in colour indicates a negative result.

3.5.6 Citrate test

The culture was inoculated in the tubes containing citrate test agar media, one control was

also maintained. The inoculated tubes were incubated at 370C, for 24 hours. The tubes were

then observed for the change in colour of the media. A change in colour from green to blue

indicates a positive result, while no change in colour indicates a negative result.

3.6.0 Identification of Fungi

3.6.1 Cultural Identification

The cultural characteristics of fungi species isolated from soil samples of dumpsite was

determined and compared wuth Atlas for identification of fungi species.

3.6.2 Microscopic Identification

The microscopic identification of fungi species was carried out using lactophenol cotton blue

staining as earlier described by Cheesbrough (2006). Briefly,a drop of lactophenol blue was

placed in a clean grease free glass slide and a small portion of the fungi myecilia were picked

using a sterile forcep and emulsified in a drop of lactophenol blue and cocered with cover

slide and examined using ×10 and ×40 objectives. The morphological characteristics of the

fungi seen under the microscope were compared with Atlas for identification of fungi.

11
 CHAPTER FOUR

RESULTS

The results showing the cultural and morphological characteristics of the isolates are shown

in Table 1. From the results, five rod-shaped and one cocci bacteria were isolated, of which

two rods were gram positive while three was gram negative. Only one cocci was isolated

which was a Gram positive bacteria.

The results of the biochemical characteristics of the bacteria isolated are outlined in Table 2.

The properties of these isolates with respects to their responses to Catalase, Oxidase, Indole,

Citrate, Urease, Methyl red, Voges-Proskauer, Glucose, Lactose, Fructose, Mannose and

Mannitol fermentation tests are shown in Table 2.

Table 3, shows the cultural and morphological characteristics of fungi species isolated from

the dumpsites. From the results, the fungi isolates Aspergillus niger, Aspergillus flavus,

Mucor sp, Penicillium sp and Trihoderma viridae: belonging to four genera (Aspergillus,

Mucor, Penicillium and Trichoderma) .

The results of frequency of occurrence of bacteria isolates from soil of dumpsite is outlined in

Table 4. The bacteria species with the highest frequency of occurrence are Bacillus sp and

Pseudomonas aeroginosa(100%), followed by Pseudomonas flourescens (60%), two bacteria

species have the lower frequency (40%) Micrococcus sp and Serratia sp and Proteus

mirabilis has the lowest.

Table 5, shows the results of frequency of occurrence of fungi isolates. The fungi with the

highest frequency of occurrence were Aspergillus niger, Aspergillus flavus and Mucor sp

(100%). The fungi isolates with the lowest frequency of occurrence with (40%) each were

Trichoderma viridae and Penicillium sp.

12
 Table 1: Cultural and Morphological Characteristics of Bacteria Isolated from soil of
Dumpsites in Keffi Metropolis, Nigeria.

HAPE SURFACE OPACITY/COLOR CONSISTENCY ELEVATION EDGE PIGMENTATION GRAM MO

STAIN
egular Dry Whitish translucent Brittle/ Flat Convex Non Positive
non- mucoid
egular Smooth Light red/ Non- mucoid Raised convex Orange-red Positive
translucent
egular Rough Light blue/ Non- mucoid Flat Convex Blue- green Negative
translucent
egular Rough Light greenish- Non- mucoid Flat Convex Greenish- yellow
yellow/ translucent Negative
egular Smooth Whitish/ Non- mucoid Raised Convex Nil Positive
translucent
egular Swarming Whitish/ Non- mucoid Flat Convex Nil Negative
translucent

13
 Table 2: Biochemical Characteristics of Bacteria Culture Isolated from soil of
Dumpsites in Keffi Metropolis, Nigeria.

CATALASE OXIDASE INDOLE CITRATE UREASE METHYL V- GLUCOSE LACTOSE

RED P
+ + - + - - - + +
+ + + - - + - + -
+ + - + - - - -
-
+ + = + - - - + -

+ + + - - + - + +

+ + - + + - + + -

means Positive - means Negative Spp means Specie

14
Table 3: Cultural and Microscopic Characterization of Fungi Species Isolated from Soil
of Dumpsite in Keffi Metropolis, Nigeria.

SURFACE REVERSE INFERENCE

Black with whitemyecilia Grey Aspergillus niger
Pale green Green Aspergillus fumigatus
Yellow- green Brown Aspergillus flavus
White Pink Mucor sp
Dark green Dark brown Trichodermaviridae
Pale- green Pale- brown Penicillum sp

15
Table 4: Frequency of Occurrence of Bacteria Species Isolated from soil of Dumpsites in
Keffi Metropolis, Nigeria.

Bacteria Isolates Location

No( )
A/L H/C D/K E/P
S/H
Bacillus Spp + + + +
+ 10(100)

Micrococcus Spp + - + -
- 2(40)

Pseudomonas aeroginosa + + + +
+ 10(100)

Pseudomonas fluorescens + + - +
- 3(60)

SerratiaSpp + - + -
- 2(40)
Proteus mirabils + - - -
- 1(20)

= Positive sp = species A/L –Angwan Lambu S/H –

School of Health
H/C – High court - = Negative D/K –Dadin kowa E/P –
Emir’s Palace

16
 Table 5: Frequency of Occurrence of Fungi Species Isolated from soil of
Dumpsites in Keffi Metropolis, Nigeria.

Fungi Isolates Location

No( )
A/L H/C D/K E/P
S/H
Aspergillus niger + + + +
+ 10(100)

Aspergillus flavus + + + +
+ 10(100)

Mucor Sp + + + +
+ 10(100)

Trichoderma viridae + - - +
- 2(40)

Penicillium Sp - + -
+ - 2(40)

means Positive - means Negative Sp means Specie

A/L –AngwanLambu
H/C – High court D/K –Dadinkowa E/P – Emir’s Palace
S/H – School of Health

17
 CHAPTER FIVE

DISCUSSION, CONCLUSION AND RECOMMENDATION

5.1 Discussion

The bacterial species isolated included Bacillus sp, Micrococcus sp, Proteus mirabilis,

Pseudomonas aeroginosa, Pseudomonas fluorescens and Serratia sp. The most frequently

encountered bacteria were Bacillus sp. and Pseudomonas aeroginosa while the least

encountered were Micrococcus sp, Proteus mirabilis, Pseudomonas fluorescens and Serratia

sp. The fungal isolates were Aspergillus niger, Aspergillus flavus, Mucor sp, Penicillium sp

and Trichoderma viridae: belonging to 4 genera (Aspergillus, Mucor, Penicillium and

Trichoderma) were isolated from the dump sites. . The bacterial species identified in the

present study are almost similar to those reported by Rahkonen et al. 1990; Crook et al. 1986;

Rylander et al. 1964 and Markanday et al. 2004. From the microbial study, it is evident that

most of the bacteria, which commonly occur in the air, soil, plants, food and water, were

opportunistic pathogens, which may cause infections. The most frequently isolated fungi in

the present study belonged to the genus Aspergillus. This agrees with Sharma et al. (1997)

who reported on an examination of soils from residential garbage of Bentul, India. In all the

soil samples that were analysed, only two genera, namely, Aspergillus and Mucor were most

frequently isolated. Wienrich et al.(1999). Also recommended A. fumigatus and A. niger as

leading spores for the behaviour of the total concentration of fungi in the bio-waste due to

their frequency of detection and seasonal dynamism. These two Aspergillus species were also

encountered in the present study. Aspergillus is well known for the spoilage of varieties of

food materials. Kannan et al. (1994), has earlier mentioned that Aspergillus is known to

produce aflatoxin a mycotoxin that is a toxic and carcinogenic metabolite produced by the

genera. The high amount of aflatoxins present in contaminated food exerts their toxicological

effect in animals and man. Aspergillus fumigatus is known to be associated with dust and its

18
endotoxins are found in landfills and compost plants (Clark et al., 1983). Mucor sp. was also

identified. It is the causal organism of fruit and vegetable rot, besides being responsible for

mycoses of the lungs in human beings (Wachukwu et al., 2010). There could be potential

risks to the environment and health due to the improper handling of solid wastes. The direct

health risks concern mainly the workers on the field who need to be protected as far as

possible from contact with wastes. For the general public, the main risk to health is indirect

and arises from the breeding of disease vectors primarily flies and rats.

Fungi, especially Aspergillus sp secrete mycotoxins that are poisonous to health when

contacted (Onuegbu, 2002). The isolation of Bacillus, Micrococcus, Proteus, Pseudomonas,

and Serratia species from the dumpsites is an indication that microbes are not only

ubiquitous in nature but also populate the soil, thus increase the nutritional value of the soil.

These microbes produce enzymes like DNase and Hyluronidase, among others that help

degrade waste materials at dump sites. Secretion of enzymes by fungi was reported by

(Chukwu et al. 2004). All the bacterial isolates reported in this study have been reported to be

associated with waste waste biodegradation. Bacillus, Proteus, Pseudomonas Micrococcus,

Serratia among others were reported by Ekundayo (1977); to be associated with waste.

Bacillus and Pseudomonas were also reported by Cook et al., (1964). Liu and Chen (1980)

reported Serratia. Pseudomonas has been widely reported to be associated with waste

(Sabry,1992.)

All the bacteria genera reported in this study have been reported by Cook et al (1964) and

Monica Chesborough (1985), as potential pathogens. That is, they are capable of causing

disease. Also, all the fungal genera reported in this study with the exception of Penicillium

are potential pathogens (Thomas, 1973; Manson-Bahr and Apted, 1982). Pavoni et al. (1975),

reported that truly pathogenic forms may survive in waste.

19
Nigeria should therefore direct her efforts towards the treatment of waste before disposal as

to minimize the health hazards associated with dumping of waste.

5.2 Conclusion

Municipal solid waste is a combination of different substrates thus it is an ideal enrichment

media for cultivation of numerous microorganisms. Bacteria in this environment are

metabolically active. Therefore, it is important to understand the waste derived bacteria in

ecological terms and as a resource for biotechnology. Our present study evidently revealed

that municipal waste dumpsite is a potential source for wide range of fungi. Moreover, it can

be an imperative resource for bio prospecting novel/ rare species, which could yield valuable

bioactive molecules necessary for ecofriendly degradation of waste and can act as a good

substitute in chemical processes in industry.

20
5.3 Recommendation

In order to enhance the quality of waste from dumpsites in Keffi and protect the lives of

people, the following recommendations need to be considered and implemented.

i. Further studies on the molecular characterization of bacteria and fungi species

isolated from dumpsite in Keffi metropolis, Nigeria should be carried.

ii. Further studies in the biodegradation potential of bacterial and fungi species isolated

from soil of dumpsite in Keffi metropolis, Nigeria should be carried out.

iii. The activities of these bacteria and fungi if properly harnessed can be used in future

treatment plants in Nigeria in accelerating the bio-conversion of waste compost into

organic fertilizer for use in gardening, agriculture and horticulture.

21
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 APPENDIX 1

LIST OF ABBREVIATIONS

Abbreviations Meanings

Conc. Concentration
0
C Degrees Celsius
Fig. Figure
hr Hour
mg milligram
μl microliter
NA Nutrient agar
min minute
% percentage
g gram
ml milliliter
μg microgram

26
 APPENDIX 1I

Media used for Isolation of Bacteria.

Nutrient Agar media

Components Quantity(gm/L)

Peptone 5.0

Beef extract 3.0

Sodium chloride 5.0

Agar 15.0

pH 7.0

27
 APPENDIX III

Mannitol fermentation test

Components Quantity(gm/L)

Agar 15.0

Nacl 7.5

Mannitol 1

Peptone 10

Beef extract 1.0

Phenol red 0.025

pH 7.4

28
 APPENDIX IV

MRVP Broth

Components Quantity(gm/L)

Peptone 5.0

Glucose 5.0

Dipotassium

phosphate 5.0

pH 6.9

29
 APPENDIX V

Colony Morphology:

(a) SHAPE

(b) MARGIN

c) ELEVATION

(d) TEXTURE: Hard, Brittle, Gumming

(e) SURFACE: Smooth, Rough

(f) OPACITY: Opaque, Transparent

(g) CELLULAR MORPHOLOGY

30
31