MICROBIOLOGICAL SURVEILLANCE ON THREE LANDFILL SITES IN

LAGOS STATE

BY

AFOLABI, Oyinkansola Bridget

Matric Number: 200561029

FACULTY OF SCIENCE

DEPARTMENT OF MICROBIOLOGY

LAGOS STATE UNIVERSITY

A PROJECT REPORT SUBMITTED TO THE DEPARTMENT OF

MICROBIOLOGY IN PARTIAL FULFILLMENT OF THE

REQUIREMENTS OF THE AWARD OF B.Sc. (HON'S) IN

MICROBIOLOGY

JULY, 2024

i
 CERTIFICATION

This is to certify that AFOLABI, Oyinkansola Bridget of the Department of Microbiology,

Lagos State University with Matriculation Number: 200561029, successfully completed this

undergraduate research project under my supervision.

__________________ ___________________

DR. O.A. OJO-OMONIYI DATE

Supervisor

__________________ ____________________

DR. A. A. ANJORIN DATE

Head of Department

ii
 DEDICATION

This Project research report is dedicated to the Almighty God, for his privilege, guidance and

protection and also to my beloved Parents and others for their moral support and encouragements.

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 ACKNOWLEDGEMENT

All praises to the Almighty God for his mercies and grace granted unto me to be strong, to

understand and carryout this project research successfully.

I, shall forever be grateful to my Parents Mr. and Mrs. Afolabi and my amiable supervisor Dr.

Ojo-Omoniyi.

My immense gratitude and appreciation go to everybody in the department of Microbiology and

my fellow course mates, thank you very much.

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 TABLE OF CONTENTS

TITLE PAGE

CERTIFICATION

DEDICATION

ACKNOWLEDGMENT

TABLE OF CONTENTS

LIST OF TABLES

LIST OF FIGURES

ABSTRACT

CHAPTER ONE

INTRODUCTION AND LITERATURE REVIEW

1.1 INTRODUCTION

1.2 LITERATURE REVIEW

1.2.1 Solid Waste Management

1.2.2 Sources, Composition and Characterization of solid waste

1.2.3 Waste generation and Management in Nigeria

1.2.4 Waste management and disposal

1.2.4.1 Use of microorganisms in waste management

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 1.2.5 Plastic waste management

1.2.5.1 Types of Plastic

1.2.5.2 Negative Effects of Plastic Waste

1.2.5.3 Microbial Degradation of Plastic

1.4 Statement of Problem

1.5 Aim of Study

1.6 Objectives of Study

CHAPTER TWO

MATERIALS AND METHODS

2.1 Laboratory and Study Area

2.2 Sample Collection

2.3 Sample Processing

2.4 Materials used

2.5 Equipment used

2.6 Reagent used

2.7 Glassware sterilization

2.8 Media used

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 2.8.1 Nutrient Agar (NA)

2.8.2 Sabouraud Dextrose Agar (SDA)

2.9 Microbial Isolation

CHAPTER THREE

RESULTS

CHAPTER FOUR

DISCUSSION

CONCLUSION

RECOMMENDATION

REFERENCES

APPENDIX

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 LIST OF TABLES

Table 1: Waste generation and density in major cities in Nigeria.

Table 2: Plastic degrading fungi

viii
 LIST OF FIGURES

Figure 1: A general Composition and classification (by material) of Municipal Solid waste

Figure 2: Municipal solid waste generation in some African countries.

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ABSTRACT

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 CHAPTER ONE

INTRODUCTION AND LITERATURE REVIEW

1.1 INTRODUCTION

Wastes are substances produced in our daily (consumption, recreational, production and living)

activities, which are unwanted and are no longer useful to us. They are those materials, which are

generated as a result of normal operations over which we have control in terms of their

production, disposal or discharge. The problem of waste management is rapidly assuming

enormous proportions, and it is a problem intensified by our “throwaway” society (Taiwo, 2009).

Waste if not properly treated and handled, not only threatens human life in the short term, but the

environment as a whole in the long run (Ogwueleka, 2009). Some of the common waste disposal

techniques in some countries such as waste dumps, landfills and incinerator have proved

inadequate (Uchegbu, 2002), and this has contributed to the pollution of the environment. Waste

disposal is one of the major problems being faced by all the nations across the globe and that is

why it is everyone’s business since it is produced on a daily basis (Ogunji et al., 2004).

Solid waste (SW) is the waste produced in a community by household, institutional and/or

commercial activities with the exception of industrial and agricultural wastes. Hence, SW

includes residential, commercial and institutional wastes which are claimed to be non-hazardous.

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

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

market places and other recreational areas. Local and regional factors, contribute to the

1
dissimilarities in SW composition, such as climate and level of commercial activity. In non-

developed countries the content of organic matter in SW is found to be higher due to the rapid use

2
of fresh and unprocessed vegetables. Hence, they have the potential to be converted into organic

fertilizer for farm use because these are bio-degradable in nature. On account of these about 53%

of waste composition are the biodegradable fraction (paper, garbages, garden and food wastes).

Therefore, the biodegradation of these wastes forms an important factor of an integrated solid

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

needs 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 (Mohapatra, 2006; Amalraj, et al., 2006).

In developing countries like Kenya Solid Waste Management (SWM) faces lots of challenges due

to weak economies, poor administrative capacities and inabilities to enforce environmental

legislation. In most developing countries, the SWM is of serious matter especially due to rapid

urbanization (Khajuria et al., 2008).

Daily solid waste formation per capita ranges from 2.75 to 4.0 Kg in high income countries and in

countries with low incomes is 0.5 to 0.8 Kg (Zurburgg, 2002). Nairobi generates solid waste on

daily basis about 4,000 tons while in Kisumu, about 400 tons in everyday (Waston, 2009; Munala

and Moirongo, 2011). Dandora dumping site has been documented to negatively affect the health

of thousands of Nairobi residents which covering an area of about 26 hectares. Besides

composting, other SWM practices include combustion/ incineration, landfills, source reduction,

and recycling. Composting is more desirable because it is cheap, prevents emissions of

greenhouse gases, saves energy, reduces pollutants, and conserves resource. With this it can also

supply valuable raw materials for agriculture and reduce the need for new landfills and

combustors (Colon and Fawcett, 2006).

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1.2 LITERATURE REVIEW

1.2.1 Solid Waste Management

Solid waste management issue is the biggest challenge to the authorities of both small and large

cities’ in developing countries. This is mainly due to the increasing generation of such solid waste

and the burden posed on the municipal budget. In addition to the high costs, the solid waste

management is associated lack of understanding over different factors that affect the entire

handling system. An analysis of literature and reported related to waste management in

developing countries, showed that few articles supplied quantitative information. The objective of

the mentioned studies was to determine the stakeholders’ action/behavior that have a role in the

solid waste management and to analyze different factors that affect the system. The studies

carried out in 4 continents, in 22 developing countries and on more than thirty urban areas. A

combination of variable methods that were used in this study was mentioned in details in order to

encourage the stake-holders and to assess the factors influencing the performance of the solid

waste management in the studied cities (Guerrero et al., 2013).

Population increases, rapid urbanization, booming economy, and the rise in the standard of living

in developing countries have greatly accelerated the rate, amount and quality of the municipal

solid waste generation (Minghua et al., 2009).

1.2.2 Sources, Composition and Characterization of solid waste

Municipal solid waste (MSW) is one of the important challenges to the environment.

Municipalities; generally; are responsible for the waste management. They have to provide an

effective and efficient system to the inhabitants. Nevertheless, they are; often; facing with many

4
problems beyond the ability of the municipal authority to handle the MSW. This is essentially due

to financial resources, lack of organization and complexity (Burntley, 2007).

The composition of MSW varies significantly from one municipality to another and from country

to country significantly. Such variation depends mainly on the life style, economic situation,

waste management regulations and industrial structure. The quantity and the composition of the

municipal solid waste are critical for the determination of the appropriate handling and

management of these wastes. Such information is essential and useful to put up the solid waste to

energy conversion facility within the municipality. Based on the calorific value and the elemental

composition of MSW the engineers and scientists can decide upon its utility as a fuel. Meanwhile,

such information will help in predicting the makeup of gaseous emissions. Thereafter, this MSW

is subjected to the energy conversion technologies including gasification, incineration etc.

However, the possible hazardous substances occurring in the ash should be considered carefully

(ASME, 2014). In this respect, the composition of the waste will provide valuable information on

the utility of the material for either composting or for biogas production as fuel via biological

conversion (Kumar et al., 2010).

Meanwhile, the time has a great effect on the composition of MSW. Biodegradation of such

MSW according to the time is an important factor that governs the amount of recyclable material

particularly the organic contents. The EPA estimated the amount of MSW generation in the

United States with 254 million tons in 2013. The composition and classification by material of

such MSW is given in Figure 1.

5
Figure 1: A general Composition and classification (by material) of Municipal Solid waste

Source: (U.S. EPA, 2013).

6
Household or municipal wastes are usually generated from variable sources where different

human activities are encountered. Several studies reported that the municipal solid waste that are

generated from the developing countries are mainly from house=holds (55–80%), followed by

market or commercial areas (10– 30%). The later consists of variable quantities generated from

industries, streets, institutions and many others (Nabegu and Hum, 2010). Generally, solid waste

from such sources is highly; heterogeneous in nature. Thus, they have variable physical and

chemical characteristics depending on their original sources. Their composition is yard waste,

food waste, plastics, wood, metals, papers, rubbers, leather, batteries, inert materials, textiles,

paint containers, demolishing and construction materials as well as many others that would be

difficult to classify. The heterogeneity of such generated solid waste is the major setback in

sorting and its utilization as material. Therefore, there is a proper need for fractionation and

sorting of these wastes before any meaningful treatment process. Sorting and separating of such

wastes are one of the most important and traditional methods as essential steps in solid waste

management to provide data on the quality of the separated fractions for any potential utilization.

Nevertheless, the success of any designed for solid waste segregation depends mainly on the

public awareness and the active participation of such waste generators in the different

communities (i.e., how they follow the fundamental and principles of waste sorting and

separation) (Valkenburg et al., 2008).

Waste generation is the first materials of waste management processes. It is a precondition to any

waste management plan to have sufficient knowledge of the generators of waste, its physical and

chemical attributes. The waste features vary not only from city to city but even within the same

city, as it depends on components such as the nature of local activities, food habits, cultural

traditions, socio-economic factors, season and climatic conditions. The physical and chemical

7
features aid in determining the desired frequency of collection of wastes, necessary precautions to

be taken during transportation, and methods of processing and disposal. Landfill waste is diverse

in nature and biological degradation takes place within the landfill in microenvironments. About

40 percent of the total mass paper and paper-related products are the biggest constituents in

MSW.

Solid waste generation (SWG) is a problematic and is an issue of concern everywhere in the

world, particularly in all urban centers. Such SWG is considered one of the most challenging

issues faced by most developing countries that suffer from sever environmental pollution

problems caused by the large quantities of SWG (Al-Khatib et al., 2010). Increased generation of

solid waste in urban cities affected dramatically on the sanitary related problems and the basic

services such as sanitation facilities, water supply, waste management, and transport infrastructure

(Liyala, 2011).

Several studies showed that collection, storage, transportation and final disposal of solid wastes

are a major problem in urban cities and areas (Okot-Okumu and Nyenje, 2011). Cities in East and

North Africa as well as most developing countries are also facing the same serious problems

related to SWG. The main reason of these problems is attributed to the poor economy of these

areas which accounts for the low achievement in solid waste management. Most of these

developing countries fail the in solid waste management and issue due to the limited available

resources and the competing priorities over their resources. Thus, the SWG is; indeed; one of the

serious and major problems faced by many cities in the world.

Meanwhile, the SWG and composition influenced by other socioeconomic factors including the

average family size, number of room(s), monthly income, and employment status (Sankoh et al.,

8
2012). It was also reported that there is a direct relation between the solid waste composition and

the social activities in the community (Gidarakos et al., 2006). In addition, other factors including

change in the source-sorting behavior and consumption of goods are among other factors

affecting the composition of the solid waste and the quantity in house-holds (Dahlen, 2008).

Socio-cultural, economic, legal, political and environmental factors as well as the available

resources are the main issues that affect the MSW management in all countries. That is why

adoption of any new technology for MSW management and SWG should take into account the

effect and the influence on the socio-cultural and the economy of the community.

As the result of the changes in consumption behaviors of people as well as the rapid advances of

technology, amounts and the composition of MSW have been also changed. In a study carried out

by the European Environmental Agency (2013) to study the per capita annual MSW produced by

32 European countries during 2001– 2010, they found that this waste increased in 21 countries,

and was decreased in 11 countries. The study also studied the number of wastes of 26 countries

between 2001 and 2008; they found that these amounts decreased in 6 countries (EPA, 2013).

Thus, the amounts and characteristics of the wastes varied from country to another country, as

well as from region to region even within the same city according the mentioned factors including

the usage habits of people (Khan et al., 2016).

Food Solid Waste

Sustainable and important source for certain industrially chemicals can be obtained from the large

amounts of the generated wastes in the world. Food residues and waste such as kitchen refuse,

garbage and swill (2006) are described; generally; the by product and as solid wastes of food.

Such wastes are produced from the processing, cooking, distribution, production, and

9
consumption of food. However, food wastes and their definition are greatly varied from cities and

countries to other cities and countries. Food wastes; in the European Union; are defined as ‘‘raw

or cooked of any food substances that are discarded, or intended or required to be discarded”. On

other hand, the (EPA) U.S. Environmental Protection Agency defines the food wastes as ‘‘Un-

eaten foods and food preparation wastes from residences and commercial establishments

including restaurants, grocery stores, and produce stands, institutional cafeterias and kitchens, as

well as industrial sources such as employee lunchrooms. Furthermore, ‘‘Food loss” and ‘‘Food

waste”, are recognized differently. The term ‘‘Food losses” refers to the decrease in food quality

and/or quantity. On the other hand, the term ‘‘food waste” refers to the food losses due to

retailers’ and /or consumers’ behavior (Russ and Meyer-Pittroff, 2004). However, food wastes

include the uncooked raw materials, wasted foodstuffs, and also the edible materials from

groceries or the wet market.

1.2.3 Waste generation and Management in Nigeria

Solid wastes could be defined as non-liquid and nongaseous products of human activities,

regarded as being useless. It could take the forms of refuse, garbage and sludge (Babayemi and

Dauda, 2009). The quantity and rate of solid waste generation in an area depends on the

population, socio-economic status of the citizens and the kinds of commercial activities

predominant in the area (Amori et al., 2013). Table 1 shows the distribution of waste generation

and density in some major cities in Nigeria, while figure 1 shows the municipal waste generation

in some African countries, including Nigeria.

In developing countries, open dumping of solid wastes into wetlands, watercourses, drains and

burrow pit is a prevalent form of disposal. This practice has sometimes resulted in the littering of

10
the surroundings, creates eyesore and odor nuisance. Sangodoyin19 stated that open dumping of

wastes serves as breeding place for flies, insects and rats (Ihuoma, 2012).

Table 1: Waste generation and density in major cities in Nigeria.

Lagos, Kano and Port-Harcourt are the three densely populated and most active commercial cities

in the South-western, Northern and Eastern Nigeria respectively. It is therefore expected that the

quantity of waste generation in these cities would be high. Ibadan is another commercial city in

the south-western Nigeria, ranking after Lagos.

Volume/capital/day
City Tons per month Density (Kg/m3)
(m3)

Lagos 255,556 294 0.63

Kano 156,676 290 0.56

Ibadan 135,391 330 0.51

Kaduna 114,433 320 0.58

Port-Harcourt 117,825 300 0.60

Makurdi 24,242 340 0.48

Onitsha 84,137 3770 0.53

Nsukka 12,000 310310 0.44

Abuja 14,785 280280 0.66

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 Source: (Ogwueleka, 2009).

Figure 2: Municipal solid waste generation in some African countries.

Nigeria and South-Africa are both known to be the largest economies in Africa, while Nigeria is

said to be the most populated nation in Africa. Egypt is a more populated country than South-

Africa. It is therefore not surprising that municipal waste generation in these African countries is

high.

Source: (Adelena, 2016).

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Also, commercial areas like market places commonly exhibits mountains of open refuse by the

roadside and other open location. The heaps of refuse provide excellent breeding grounds for

vectors of communicable diseases including rodents, insects, which increases the potential for the

spread of infectious diseases. It is also acknowledged that many of the diseases that affect

Nigerians, including malaria, tuberculosis and diarrhea are due to unhealthy environmental

conditions. They may also pose fire hazards apart from being eyesores and sources of unpleasant

odors. Very frequently, refuse is dumped in drainages or canals and along watercourses with

impunity. The unsanitary mode of wastes disposal, such as open urination, defecation in streams

and the dumping of refuse in pits, rivers and drainage channels are widespread and the resultant

contamination of the environment contributes to environmental degradation (Abejegah et al.,

2013).

Additionally, the volume of solid waste being generated continues to increase at a faster rate than

the ability of the agencies to improve on the financial and technical resources needed to parallel

this growth. Solid waste management in Nigeria is characterized by inefficient collection

methods, insufficient coverage of the collection system and improper disposal of solid waste. The

quantity of solid waste generated in urban areas in industrialized countries is higher than in

developing countries; though municipal solid waste management remains inadequate in the latter.

Solid waste in developing countries differs from developed countries. Most developing countries

have solid waste management problems different from those found in industrialized countries in

13
areas of composition, density, political, and economic framework, waste amount, access to waste

for collection, awareness and attitude (Ogwueleka, 2009).

On the other hand, wastewater is generated from domestic sewage, agricultural processes and

industrial effluent. Trivedi et al., (2005) stated that out of the total wastewater generated, 90.62%

find its destination into the coastal waters without any treatment. The average domestic sewage

contains organic matter, nitrogen and phosphorus, suspended solids, dissolved oxygen and

bacterial parameter (fecal coliform).

Recently, an increasing category of waste stream in the world (including Nigeria) is the Waste

Electrical and Electronic Equipment or E-waste (Ogungbuyi et al., 2012). It is a term for

electronic products that have become unwanted, non-working or obsolete, and have essentially

reached the end of their useful life. In developed countries, it equals 1% of total solid waste on an

average, while in developing countries, it ranges from 0.01% to 1% of the total municipal solid

waste generation. In Nigeria though annual generation per capita is less than 1 kg, it is growing at

an exponential pace in addition to the ever-growing hazardous waste stream. Heavy metals such

as silicon, lead, mercury, and other related items had been found in rivers, lakes and water with

adverse effects on human cells, which had been traced to illegal and indiscriminate dumping of e-

waste into water bodies thereby percolating into soil. An estimated 53,600 metric tons of e- waste

are dumped annually at Lagos state landfills which include 860,000computers, 530,000 printers,

900,000 monitors and 480,000 television sets (Okenwa, 2010; LASEPA, 2015).

1.2.4 Waste management and disposal

Waste management is the collection, transport, processing or disposal, managing and monitoring

of waste materials to minimize its consequences on humans and environment. Solid waste

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treatment techniques act to reduce the volume and toxicity of solid waste, transforming it into a

more convenient and/or beneficial form. In Awosusi, (2010) waste management is viewed as a

process of source reduction, refuse recycling, controlled combustion and controlled landfill;

energy generation from waste (energy recovery) and lastly, solid waste disposal, if the

aforementioned do not offer appropriate solution. A number of processes are involved in

effectively managing solid waste. These include monitoring, collection, transport, processing,

recycling, incineration, landfilling and compostin (Atalia et al., 2015). This includes different

types of methods such as follows thermal treatment (whereby the process use heat to treat waste

materials) such as incineration, gasification and pyrolysis, and open burning; dumps and landfills

such as sanitary landfills, controlled dumps and bioreactor landfills; biological waste treatment

such as composting and anaerobic digestion (LeBlanc, 2016).

1.2.4.1 Use of microorganisms in waste management

The microorganisms which include bacteria, fungi, algae, protozoa, and other higher animals

settle in the aerobic biological treatment systems. In a particular industrial waste disposal system,

the growth of all types of microorganisms will depend upon the chemical features of the industrial

waste, the environmental restrictions of the particular waste system and the biochemical

characteristics of the microorganisms. All of the microorganisms contribute to its over-all

characteristics, both good and bad that grow in a specific industrial waste disposal system. It is

important to identify the contributions to the over-all stabilization made by each type of organism

of the organic wastes if the waste treatment system is to be perfectly designed and performed for

highest potency (Metin et al., 2003). The growth of any or all types of microorganisms in a given

industrial waste disposal system will depend upon the chemical characteristics of the industrial

waste, the environmental limitations of the particular waste system and the biochemical

15
characteristics of the microorganisms. All of the microorganisms which grow in a given industrial

waste disposal system contribute to its over-all characteristics, both good and bad. It is important

to recognize the contributions made by each type of organism to the over-all stabilization of the

organic wastes if the waste treatment system is to be properly designed and operated for

maximum efficiency.

Bacteria:

The bacteria are the basic biological units in aerobic waste treatment systems. The diverse

biochemical nature of bacteria makes it possible for them to metabolize most, if not all, organic

compounds found in industrial wastes. Obligate aerobes. and facultative bacteria are found in all

aerobic waste treatment systems. Growth of any particular species is dependent upon its

competitive ability to obtain a share of the available organic material in the system. Bacterial

predomination will normally divide itself into two major groups: the bacteria utilizing the organic

compounds in the waste, and the bacteria utilizing the lysed products of the first group of bacteria.

The bacteria utilizing the organic compounds in the waste are the most important group and will

determine the characteristics of the treatment system. The species with the fastest growing rate

and the ability to utilize the majority of the organic matter will predominate. The extent of

secondary predomination will depend upon the length of starvation. Depletion of the organic

substrate is followed-by death and lysis of the predominate bacteria. Release of the cellular

components of the bacteria permits other bacteria to grow up. Since all biological treatment

systems are normally overdesigned as a safety factor, secondary predomination will occur. Aside

from the metabolic characteristics of the bacteria, the most important characteristic is their ability

to flocculate. All of the aerobic biological waste treatment systems depend upon the flocculation

of the microorganisms and their separation from the liquid phase for complete stabilization.

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It was first thought that flocculation was caused by a single bacterial species, Zoogloea ramigeria,

but recent studies have shown that there are many different bacteria which have the ability to

flocculate. It has been postulated that all bacteria have the ability to flocculate under certain

environmental conditions. The prime factors affecting flocculation are the surface charges of the

bacteria and their energy level. The electrical surface charge on bacteria grown in dilute organic

waste systems has been shown to be below the critical charge for auto-agglutination, 0.020 volts.

This means that Brownian movement provides sufficient energy to overcome the repelling

electrical forces when two bacteria approach each other and to permit the Van der Waal forces of

attraction to predominate and hold the two bacteria together. Autoagglutination does not take

place if the energy level of the system is sufficiently high to permit the bacteria to multiply and to

be rapidly motile. Autoagglutination, or flocculation, occurs only after the bacteria lack the

energy of motility to overcome the Van der Waal forces. Once floc has started to form, some of

the bacteria die and lyse. An insoluble fraction of the bacterial cell is left which is primarily

polysaccharide. The older the floc becomes; the more polysaccharide builds up and the less active

bacteria are entrained in it.

Fungi:

Fungi play a vital role in the stabilization of organic wastes. The fungi can metabolize almost

every type of organic substances found in industrial wastes like the bacteria, The fungi have the

potentiality to predominate over the bacteria but they do not include under unusual environmental

conditions. The filamentous fungi found in industrial wastes makes them unpleasant since they do

not form a tight dense loc and settle easily. The filamentous fungi predominate over the bacteria

at low pH, at low oxygen tensions and at low nitrogen. Metabolism does not proceed to carbon

dioxide and water under reduced oxygen levels, but stops with the formation of aldehydes,

17
organic alcohols, and acids. If the system needs adequate buffer, the organic acids weaken the pH

to the more favourable range for fungi. Thus, it can be visualizing that low oxygen tension and pH

can be interrelated. Most of fungi grow at pH 4 to 5 while some bacteria are allowed to grow well

sufficient to compete. Fungi need less nitrogen than bacteria per unit mass of protoplasm. The

fungi are able to integrate in nitrogen deficient wastes, more active masses of protoplasm than the

bacteria from the wastes and predominate. Bacteria average 10 to 12% nitrogen while fungi range

from 5 to 6% nitrogen approximately. Fungi will be present and will aid in the stabilization of the

organic matter under normal environmental conditions. But the fungi are of secondary importance

and will not predominate. Hence, microorganisms are vital to humans and the environment, as

they participate in the nitrogen and carbon cycles, as well as fulfilling other vital roles like

recycling other organisms' dead remains and waste products through decomposition.

Microorganisms also have an important place in higher multicellular organisms as symbionts.

Protozoa:

The protozoa are the simplest animals found in waste disposal systems. The role that the protozoa

play in stabilizing organic wastes has only recently been clarified by combining a study of pure

culture protozoa with the natural observations in various biological treatment systems. This study

showed that rather than being the primary mechanism of purification, the protozoa were

responsible for reducing the number of free-swimming bacteria, thus aiding in producing a

clarified effluent. The succession of protozoa had long been observed in biological waste disposal

systems but there was no explanation of the reasons for this succession. The succession of

protozoa is affected by the same factors which affect the predomination of any biological species.

The type of food and the competition for food are the major factors which determine the

predomination of the protozoa. The Sarcodina are only briefly found in aerobic waste treatment

18
systems since they do not find sufficient food to compete with the bacteria and other biological

forms. The Phyto-Mastigophora survive a little longer than the Sarcodina as they take in soluble

organics for their food but they are unable to compete against the bacteria and are soon displaced.

The Zoo-Mastigophora predominates over the Phyto-Mastigophora in that they are able to utilize

the bacteria for food rather than compete with the bacteria for food. But the Zoo-Mastigophora

give way to the free- swimming Ciliata which have a better mechanism for obtaining the bacteria

and other food components. As the system becomes more stable, there are less and less free-

swimming Ciliata. The low-energy-requiring stalked Ciliata displace the high-energy-requiring

free-swimming Ciliata. But soon the system becomes so stable that the stalked Ciliata cannot

obtain enough energy and die out of the system.

The succession of protozoa offers a good index of stability of the biological waste treatment

system. Efforts have been made to relate the numbers of protozoa to the degree of stabilization

but they have not been successful since the same numerical population exists at two separate and

distinct levels of purification. Low numbers of free- swimming Ciliata occur at both a low degree

of purification, 20 to 40%, and at high purification, 75 to 95%. The relative types of protozoa and

relative numbers can be used for any particular system to estimate the rough efficiency, ±10%, of

any biological treatment system. The protozoa have more complex metabolic systems than do

bacteria or fungi which make the protozoa more sensitive to toxic organic compounds. In systems

containing toxic organic compounds, regular observations of the protozoa can be used as an

indicator of the toxic concentration and to warn of potential toxicity to the bacteria which are

responsible for stabilization of the wastes. The protozoa can also be used to indicate deficiencies

of certain essential elements such as nitrogen or phosphorus. Nutrient deficiencies will reduce

both number of species and number of any particular species.

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Algae:

The algae are the third form of biological plants which play a part in the over-all stabilization of

organic wastes. Since the algae obtain their energy for synthesis from sunlight, they do not have

to metabolize the organic compounds like the bacteria and the fungi. To form protoplasm the

algae primarily utilize the inorganic components of the wastes, for example, ammonia, carbon

dioxide, phosphate, magnesium, potassium, iron, calcium, sulfate, sodium and other ions. It is

possible to have algae and the bacteria predominate together since they do not utilize the same

waste components. The bacteria metabolize the organic components of the waste and release

some of the inorganic components utilized by the algae. During protoplasm synthesis the algae

release oxygen which is taken by the bacteria to bring about complete aerobic stabilization of the

organic matter. In the absence of sunlight, the algae must obtain the energy required to stay alive

from the metabolism of organic matter in the same manner as bacteria and fungi. This organic

matter normally comes from stored food within the cell but in some algal species it can come

from the organic material in the

Viruses:

These are particles assembled from the biopolymers, which are capable of multiplying and

assembling as new virus particles inside living prokaryotic or eukaryotic cells. In the

environment, viruses are important for the following reasons: pathogenic viruses must be

removed, retained or destroyed during water and wastewater treatment; viruses of bacteria

(bacteriophages) can infect and degrade the bacterial cultures in the environment; and

bacteriophages can be used for the detection of specific microbial pollution of waste in the

environment (Ivanov, 2010)

20
1.2.5 Plastic waste management

Plastics are relatively inexpensive, lightweight, resistant to water, and strong products can be used

for many purposes. This polymer is widely used in almost all fields of life. The use of plastic

products is likely to increase by 9% every year (Thompson et al., 2009). World plastic production

reached 400 million tons annually (UNEP 2018). Unfortunately, plastic contains various toxic

components hazardous to living things. The massive use of plastic products leads to plastics

accumulation in the environment (Thompson et al., 2009), because these pollutants require a very

long time to be completely degraded in nature, thus pollute the water, land, and air (Kathiresan

2003, Soud 2019).

Plastic wastes have become contaminants on land and in the aquatic environment. Thus, effective

plastic waste processing methods are needed (Mandan and Arya 2017). The common methods

used in processing plastic wastes are landfill, incineration, and recycling. Each of these methods

has its weaknesses. Thus, they are not effective in solving plastics pollution. Landfill plastic

wastes processing requires a long time without rotting. Incineration treatment will produce toxic

gases into the environment (Al-Salem et al., 2009, Hopewell et al., 2009, Gan and Zhang 2019).

The incineration process of PVC, PET, PS, and PE wastes produces carcinogenic substances as

PAHs, nitro-PAHs, dioxins, and others (Al-Salem et al., 2009, Yang et al., 2018). Additionally,

recycling plastic wastes is also relatively expensive (Al-Salem et al., 2009, Hopewell et al., 2009,

Gan and Zhang 2019).

1.2.5.1 Types of Plastic

Plastics can be divided into thermoplastics and thermosets. Thermoplastic is a group of plastics

that can be remelted and reprocessed back into a product as well as recyclable. Meanwhile,

21
thermosets or thermosetting is a plastic group that cannot be remelted because the molecular

bonds contained in that plastic polymers are tightly bound in crosslink (Albano et al., 2009, Choi

et al., 2009, Bărbută et al., 2010, Choi et al., 2005, Mohammadian and Haghi 2013). The four

most commonly used plastic polymers in daily life are high-density polyethylene (HDPE), low-

density polyethylene (LDPE), polypropylene (PP), and polyethylene terephthalate (PET). They

are the most common sources of plastic pollutants that are mainly used as plastic bags (Drzyzga

and Prieto 2018). Polyethylene (PE) is the most widely used plastic. The use of that polymer

reached 29.6% of the total daily plastic used, followed by polypropylene (PP) as much as 18.9%,

polyvinyl chloride (PVC) 10.4%. Meanwhile, the use of polyurethane (PUR) reached 7.4%,

polystyrene (PS) reached 7.1% and the use of PET reached 6.9% (Wu et al., 2017, Yang et al.,

2018).

Polyethylene (PE) is a synthetic plastic that has a high molecular weight, complex three-

dimensional structure, and hydrophobic nature (Hadad et al., 2005, Shah 2009). Polyethylene is

composed of stable polymers containing bonds composed of ethylene monomers (Alshehrei

2017). The stability of the polymers makes this plastic resistant to degradation in nature (Nandi

and Joshi 2013). PP is a synthetic plastic composed of a heat-and-chemical-resistant polymer.

Because of this property, PP is resistant to degradation (Khoironi et al., 2019).

PET has two hydroxyls (OH) and dicarboxylic aromatic acids consisting of aromatic carbon rings

and two carboxyls (CO2H) (Venkatachalam et al., 2012, Farzi et al., 2019, Webb et al., 2013).

This plastic is synthesized by involving two chemical reactions (Awaja and Pavel 2005, Webb et

al., 2013). The aromatic components contained in PET caused this synthetic polymer difficult to

be degraded naturally in the environment (Webb et al., 2013).

22
1.2.5.2 Negative Effects of Plastic Waste

The massive use of plastics generates the accumulation of plastic wastes in the environment

(Ahsan et al., 2016). Plastic wastes cause many diseases for living beings, including humans.

Plastic polymers accidentally ingested lead to immune system disorders, inhibit and disturb

enzyme activities, and cause hormonal system disorders resulting in abnormalities of the

endocrine system. The carcinogenic compounds of this synthetic polymer threaten many living

creatures both on land and in aquatic environments (Pavani and Rajeswari 2014, Munir et al.,

2018).

Plastic waste polluting the land can be imperfectly degraded by environmental factors. They cause

the washing or decomposition of harmful substances of the plastic polymers into the environment.

Hazardous substances such as heavy metals, plasticizers, stabilizers, and plastic dyes will be

released into the environment. These pollutants pollute the terrestrial environment. Furthermore,

they will be carried by the water stream then pollute the aquatic environment. In particular,

around 80% of plastic pollution in the aquatic environment originated from the land (Sheavly

2005, Alabi et al., 2019).

Plastic wastes float from one place to another. They might bring many living things to new

places, introduce some invasive species to a new aquatic environment that will compete with

indigenous species (Hasnat and Rahman 2018). Fire is another adverse effect of plastic waste that

is caused by the burning of flammable plastic waste. Not only causes many types of losses, but

the smoke produced by the burning of the plastic wastes also emits toxic gases into the air like

carbon monoxide (CO) and hydrogen monoxide (HCN) (Purwaningrum, 2016).

23
Even though plastic might be degraded in nature after hundreds or thousands of years, the

incomplete degradation of that recalcitrant polymer releases toxic fragments into the environment

thus causing many problems. The fragmented toxic compounds might be accumulated in various

living things resulting in some health disorders (Kang et al., 2019). Moreover, the accumulation

of plastic wastes pollutes the view which decreases the attractiveness of tourism objects in a

country thus adversely affecting a country's economy, mainly a country that is highly depend on

tourism as the main source of its GDP. It is because of the number of plastic wastes discomfort

and reduce the tourist’s interest toward a tourism object (UNEP 2018). After all, the complete and

environmentally safe plastic waste processing method is needed. Various studies have reported

that the degradation of plastic waste using enzymes produced by microbes such as fungi is the

appropriate method to overcome plastic pollution (Kang et al., 2019).

1.2.5.3 Microbial Degradation of Plastic

Many studies have reported the ability of fungi in plastic biodegradation (Table 2). Using fungi as

bioremediation agents is an appropriate method to decrease the number of plastic wastes polluting

the environment. The rapid growth of fungi in many substrates is a beneficial factor in

biodegradation. Because of their rapid growth, their mycelium will be spread out covering the

entire substrate surfaces then penetrating them to start the biodegradation process. Fungi can be

found in any kind of environment, including extreme environments such as low pH and arid (Kim

and Rhee 2003, Nandi and Joshi 2013). Fungi isolated from soil exposed to plastics are important

plastic wastes biodegradation agents (Nandi and Joshi 2013). There are many studies that reported

some plastic degrading fungi including Gliomastix sp., Chaetomium sp., Fusarium sp.,

Mortierella sp., and Paecilomyces sp. (Nathania and Kuswytasari 2013, Hardiyanti et al., 2017).

Indigenous fungi isolated from plastics contaminated landfill are potential agents of plastic

24
biodegradation as Trichoderma sp., Aspergillus flavus, and Aspergillus niger (Hardiyanti et al.,

2017).

There are many studies that reported polyethylene (PE) degrading fungi, Myceliophthora sp. is

one of them. This fungus produces laccase catalyzing the degrading process of plastic polymers. It

produces laccase optimally at pH 5.0 and temperature 30oC. This plastic degrading fungus can

grow on a medium containing polyethylene. Electron microscopy analysis showed that the surface

of the plastic exposed to the fungus was damaged, thus closely related to the activity of the

laccase produced by the fungus (Khalil et al., 2013).

Table 2: Plastic degrading fungi

Plastics Type Fungi Isolate Sources References

Polyethylene (PE) Nandi and Joshi

Aspergillus niger Garbage soil
(2013)

Nandi and Joshi

Aspergillus flavus Garbage soil
(2013)

Sangale et al.,
Aspergillus terreus Rhizosphere Avicenia
(2019)

Fusarium sp. AF4 Sewage sludge Shah et al., (2009)

Chaetomium Dumpsite soil Sowmya et al.,

25
 globosum (2014)

A.fumigatus Red sea Alshehrei (2017)

Aspergillus flavus Red sea Alshehrei (2017)

Low-density Aspergillus Mandan and Arya

Polythene bags
polyethylene (LDPE) japonicas (2017)

Penicillium sp. Red sea Alshehrei (2017)

Aspergillus nomius Landfill soil Munir et al., (2018)

Aspergillus Plastic waste dumping Verma and Gupta

High-density
fumigatus site (2019)
polyethylene (HDPE)

Ingavale et al.,
Aspergillus niger Culture
(2018)

Polyethylene Asmita et al.,

Aspergillus niger Culture
terephthalate (PET) (2015)

Plastic contaminated El-Morsy et al.,

Monascus ruber
soil (2017)
Polyurethane (PUR)
Isolated from
Pestalotiopsis sp. Lii et al., (2017)
Nephentes ampullaria

Polyhydroxybutyrate Alterneria alternate Culture Aburas (2016)

26
 Trichoderma sp. Culture Aburas (2016)
(PHB)
Trichoderma sp. Soil Râpă et al., (2014)

Polystyrene -
Soil, plastic debris, Ibrahim et al.,
Polyurethane A. flavus (FopI-2)
latex and plastic shield (2011)
(PS-PUR)

1.4 Statement of Problem

Landfills are essential components of waste management systems but can pose significant

environmental and health risks due to microbial contamination. In Lagos State, where

urbanization and waste production are escalating, landfills may harbor pathogenic

microorganisms that could contaminate surrounding soil, water sources, and air, affecting human

health and local ecosystems. There is a critical need to analyze the microbiological profile of

these sites, focusing on both solid waste and leachate water, to ensure the safety of the

environment and public health.

1.5 Aim of Study

The aim of this study is to conduct a comprehensive microbiological surveillance of three landfill

sites in Lagos State; Ojota, Ikorodu, and Solous, to isolate, identify and quantify pathogenic

microorganisms in both solid waste and leachate water samples.

27
1.6 Objectives of Study

1. To isolate and identify bacterial and fungal species present in solid waste and leachate water

from the three selected landfill sites.

2. To quantify the levels of microbial contamination in soil and leachate water samples from

these sites.

3. To perform molecular characterization of the isolates using PCR and sequencing techniques.

4. To identify the isolated microorganisms based on their molecular characteristics.

CHAPTER TWO

MATERIALS AND METHODS

2.1 Laboratory and Study Area

The research was conducted at the Microbiology Laboratory of Lagos State University in Ojo.

Ojo is a local government area in Lagos State, Nigeria, situated approximately 37 km west of

Lagos on the eastern section of the Trans-West African Coastal Highway. It is part of the Lagos

Metropolitan Area.

2.2 Sample Collection

Samples will be collected from three major landfill sites in Lagos State: Ojota, Ikorodu, and

Solous. Both solid waste and leachate water samples will be gathered from multiple points at each

28
site. Samples will be taken from surface and subsurface layers using sterile tools and placed in

labeled containers.

2.3 Sample Processing

Soil samples were collected from each of the 3 sites, focusing on surface and subsurface layers.

The collected soil samples were gathered at each location and stored in sterile polyethylene bags.

The samples were then transported to the laboratory in a sterile box to avoid contamination. Upon

arrival at the laboratory, samples were homogenized and prepared for analysis. Leachate water

samples were collected using sterile plastic containers, ensuring to capture samples from active

drainage points and ponded areas at the landfill sites. These containers were also transported to

the laboratory in aseptically to preserve the sample quality.

2.4 Materials used

Conical flasks, aluminum foil, bijou bottle, petri dish, cotton wool, test-tube, universal bottle,

needle and syringe, hand gloves, measuring cylinder, spirit lamp, glass slide, cover slip,

autoclave, hot air oven, inoculating loop and needle, spatula, paper tape, markers, matches.

2.5 Equipment used

Refrigerator, microscope, incubator, Autoclave, Hot air oven, weighing balance

2.6 Reagent used

Ethanol, distilled water, lactophenol blue, Gram Stains (safranin, methyl red and crystal violet),

iodine, antibiotics,

2.7 Glassware sterilization

29
All glassware, including pipettes, test tubes, conical flasks, and measuring cylinders, were

cleaned, wrapped in aluminum foil, and sterilized in an oven at 170°C for 2 hours before use.

Unless stated otherwise, all culture media were covered with cotton wool, wrapped in foil paper,

and sterilized in an autoclave at 121°C for 15 minutes.

The workbench surfaces were disinfected with cotton wool dipped in 70% ethanol before and

after use. The hockey stick was flamed before spreading inoculum on agar plates. All equipment

was thoroughly cleaned before and after use, and all microbiological work was conducted near the

flame to maintain an aseptic environment.

2.8 Media used

Nutrient Agar (NA) and Sabouraud Dextrose Agar (SDA). These media were selected based on

their suitability for cultivating a broad spectrum of bacteria and fungi, respectively, which are

anticipated to be present in landfill environments. Both media play a critical role in the

microbiological analysis of landfill sites by providing distinct environments for cultivating

bacteria and fungi.

2.8.1 Nutrient Agar (NA)

Composition:

1. Peptone: Provides nitrogen, vitamins, minerals, and amino acids essential for microbial

growth.

30
2. Beef Extract: Supplies additional nutrients, including carbohydrates, nitrogenous compounds,

and salts.

3. Agar: Solidifying agent that provides a stable surface for bacterial colony formation.

4. Sodium Chloride: Maintains osmotic balance.

Preparation of Media:

1. The appropriate amount of nutrient agar powder is weighed and dissolved in distilled water

according to the manufacturer's instructions.

2. The solution is heated until the agar is completely dissolved.

3. The medium is autoclaved at 121°C for 15 minutes to ensure sterility.

4. Once cooled to about 45-50°C, the medium is poured into sterile Petri dishes and allowed to

solidify

2.8.2 Sabouraud Dextrose Agar (SDA)

Composition:

1. Dextrose: Provides an energy source for fungal growth.

2. Peptone: Supplies nitrogenous nutrients to support microbial metabolism.

3. Agar: Acts as a solidifying agent.

4. Acidic pH: The medium's acidic nature (pH 5.6) suppresses bacterial growth, favoring fungal

isolation.

Preparation of Media:

31
1. The exact amount of SDA powder is measured and dissolved in distilled water following the

manufacturer's guidelines.

2. The mixture is heated to dissolve the agar completely.

3. The medium is sterilized by autoclaving at 121°C for 15 minutes.

4. After cooling to approximately 45-50°C, the medium is dispensed into sterile Petri dishes and

allowed to set.

2.9 Microbial Isolation

The process of microbial isolation begins with the preparation of serial dilutions from the

collected samples. Solid waste samples are first homogenized and then suspended in sterile

distilled water. Leachate water samples are shaken thoroughly to ensure an even distribution of

microorganisms. From these prepared samples, serial dilutions are created by transferring 1 ml of

the sample into 9 ml of sterile distilled water, mixing thoroughly, and repeating the process to

achieve a series of dilutions, typically from 10 1 to 106. For this study, the fourth dilution (104) is

selected for inoculation.

To culture the microorganisms, 0.1 ml from the 10 4 dilution is inoculated onto Nutrient Agar

(NA) for general bacterial growth and Sabouraud Dextrose Agar (SDA) for fungal growth. The

inoculum is evenly spread across the surface of the agar plates using a sterile spreader to ensure

uniform distribution. The inoculated NA plates are incubated at 37°C for 24-48 hours, while the

SDA plates are incubated at 25-30°C for 48-72 hours.

After the incubation period, colony-forming units (CFUs) on each plate are counted to quantify

the level of microbial contamination. The morphological characteristics of the colonies are

32
observed and recorded, and further biochemical tests, including catalase, oxidase, and motility

tests, are performed on isolated bacterial colonies to aid in their identification.

2.6 Identification of Isolates

2.6.1 Morphological Characteristics

2.6.2 Gram Staining

2.6.3 Molecular Identification

CHAPTER THREE

RESULT

3.1

33
CHAPTER FOUR

DISCUSSION

34
CONCLUSION

35
RECOMMENDATION

36
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