AMERICAN UNIVERSITY OF NIGERIA DEPRTMENT OF …

AMERICAN UNIVERSITY OF NIGERIA

DEPRTMENT OF NATURAL AND ENVIRONMENTAL SCIENCES

Senior Research Thesis

IDENTIFICATION AND CHARACTERIZATION OF BIOREMEDATION

POTENTIAL OF MICROORGANISMS IN SOIL FROM WASTE

DUMPS IN YOLA-JIMETA NORTHEASTERN

By

SHOM MLUMUN GRACE

A00017174

Submitted in partial fulfillment of the

requirements for the degree of

Bachelor of Science

2018

ii

AMERICAN UNIVERSITY OF NIGERIA

DEPARTMENT OF NATURAL AND ENVIRONMENTAL SCIENCES




This thesis represents my original work in accordance with the American University

of Nigeria regulations. I am solely responsible for its content.

SHOM GRACE M.

______________________________ ________________

Signature Date

I further authorize the American University of Nigeria to reproduce this thesis by

photocopying or by any other means, in total or in part, at the request of other

institutions or individuals for the purpose of scholarly research.

SHOM GRACE M.

______________________________ ________________

Signature Date

iii




SHOM GRACE M.

A00017174

Approved by

Supervisor: Dr. Hayatu Raji

Chair, Department of Natural and Environmental Sciences

______________________________ ________________

Signature Date

Research Co-supervisor: Dr. Victoria Adams

Department of Petroleum Chemistry

______________________________ ________________

Signature Date

iv

DEDICATION

This work is dedicated to God almighty, to my loving and caring parents Mr/Mrs.

Terver Shom and Mrs Ayuk Tabe.

v

ACKNOWLEDGEMENTS

All praises and thanks are goes to God almighty for being my strength throughout

this project. A very big bear hug to all my family and friends. To my awesome

friends you are the best gift life has ever given me.

To Mr. and Mrs. Terver Shom thank you for being a lovely and wonderful being.

You have been a mini god right from day one. In you, I found peace and happiness. I

love you and want you to know that I will forever respect and cherish the genuine

bond we share. Thank you for being the best parents life can offer. You are truly a

delight.

My sincere gratitude and appreciation goes to Dr. Hayatu Raji and Dr. Victoria

Adams. You are more than a faculty advisor to me and to this outstanding project.

You have been a a father and mother figure. I sincerely appreciate you. This project

and would not have been possible without you. Thank you for your unfailing

attention and for believing in me. I appreciate the many hours you spent on the

sampling technique and the many hours we spent on correcting my scattered nature.

It was a pleasure working with you.

Special thanks to Mr Stanley Bitrus and Japhet for helping me sample collection. I

also appreciate Miss Gloria Onyebarachi and Mr Mohammed for their assistance in

the NES and Petroleum Chemistry labs. Thank you so much Miss Gloria, your

sleepless nights, I cannot appreciate you enough. You are a true definition of

friendship. Lastly, I thank AUN for providing a platform for me to achieve beyond

my potential.

vi


POTENTIAL OF MICROORGANISMS IN SOIL FROM WASTE DUMPS IN

YOLA-JIMETA NORTHEASTERN

SHOM GRACE M.

American University of Nigeria, 2018

Supervisor: Dr Hayatu Raji

Chair of the Department of Natural and Environmental Sciences

Co-supervisor: Engr. (Dr.) Feyisayo Victoria Adams

Department of Petroleum Chemistry

ABSTRACT

Biodegradation has proved over time that it is the cheapest and safest method human

can use to tackle waste and population. The study of biodegradation of

polypropylene revels that Actinomycyte and three other unknown strains are capable

of biodegrading polypropylene (making new functional group) within 3weeks. The

new functional group seen after 3 weeks were ester, cyanide, and ketone. The

microbial community at the Yola waste are a community of diverse organism. Each

with its own unique morphology, and growth pattern. All organisms were gram

positive. This means that they can adapt to high stress and a resist turgor pressure.

Actinomycyte spp was able to biodegrade polypropylene by breaking the carbon to

hydrogen, and breaking down carbon hydrogen bond to make carbon oxygen bonds.

vii

TABLE OF CONTENTS

CERTIFICATION…………………………………………………………………....ii

READERS’APPROVAL……………………………………………………............iii

DEDICATION……………………………………………………………….............iv

ACKNOWLEDGEMENTS…………………………………………………..............v

ABSTRACT………………………………………………………………...............vi

LIST OF TABLES…………………………………………………………………...ix

LIST OF FIGURES…………………………………………………………............x

CHAPTER 1………………………………………………………………………….1

INTRODUCTION………………………………………………………………........1

Waste…………………………………………………………………………….........1

Composition……………………………………………………………......................2

Waste management ………….…………………………………………………………4

Bioremediation…………………………………..……………………………………5

Degradation of waste materials by microorganisms………………………………....7

Keratin degradation…………………………………………………….…………...7

Plastic degradation…………………………….........................................................8

Anaerobic digester………………………………………......………………..……..9

Types of bioremediation…………………………………………….……………....11

Compositing ………………………………………………………………………...11

Bioventing ………………………………………………..........................................12

Case of Nigeria………………… ………………………………..................................13

AIMS & OBJECTIVES…………..……………………………….………………..15

CHAPTER 2……………………………………………………………………….16

MATERIALS & METHODS……………………………………………...16

viii

Study site………………………………………………………..................16

Sampling techniques……………………………………………………….17

CHAPTER 3………………………………………………………………………..20

RESULTS…………………………………………………………………..20

CHAPTER 4………………………………………………………………………....25

DISCUSSION…………………………………………………………….…25

CHAPTER 5…………………………………………………………………………33

CONCLUSIONS AND

RECOMMENDATIONS……………………………………………………………33

.

REFERENCES…………………………………………………………………….. 34

ix

LIST OF TABLES

Table 1: IR interpretation chat for all sample after 2 week of

biodegradation……………………………………………………………………….22

Table (2). Waste dump soil organism biochemical

test…………………………………………………………………………………...23

x

LIST OF FIGURES

Fig:1 Composition of household wastes……………………………………………...2

Fig:2 Municipal waste collection data for different countries………………………..4

Fig:3 Different bioremediation techniques used across

the world………………………………………………………………...…………...13

Fig:4 Map of Yola town……………………………………………………..............16

Fig:5 IR spectrometer reading of Polypropylene (PP): B untreated (un-inoculated), A,

C and D treated samples (inoculated with strain 1 and 2 for 2weeks in a broth of PP

and water ………………………………………………………………….…….…..21

Fig:6 Colour change due to pseudomonas spp………………………….…………..23

Fig:7 Microscopic view of bacteria found in the total sample soil at total magnification

of 1000X……………………………………………………………………………..24

Fig:8 Molecular structure of polypropylene………………………………………...27

Fig:9 Molecular structure of Ester………………………………………………......28

Fig.10 Molecular structures of aldehyde and ketone..................................................28

1

CHAPTER 1

INTRODUCTION

Every year, about 1.3 billion tons of waste is generated globally. By 2025, it is

estimated that this will increase to 2.2 billion tons, with a large sum (2.13

kg/capita/day) from developing countries (Bhada-Tata, &Hoornweg, 2012). The term

waste refers to items or products (organic and inorganic) that are considered useless

or have lost their value (Bhada-Tata, & Hoornweg, 2012). It is a broad term items

such as animal bones, plastic bags and bottles, and used clothes. Waste generation

has been in existence, since the beginning of agricultural revolution, and it can be

traced back to the first human civilization. As a result, waste is inevitable and cannot

simply be avoided, due to urbanization (Muhammad, Huma, Munir, & Atiq, 2015).

Information age, urbanization and industral age has led improve human lifestyle, in

many cities, which is the major cause of increase in solid waste production (Renou,

2008).

With the emergence of the industrial age, and then the information age, urbanization

could not stifled because of the human desire for securing a more comfortable life.

This led to the production of waste products. Urbanization generally brings economic

prosperity, and higher waste production. This is because people living in cities

usually earn more income, and have several options from which they can choose

from (Renou, Givaudan, Poulain, Dirassouyan, & Moulin , 2008).However, urban

settlements are known to be densely populated, leading to greater amount of waste

compared to non-urban areas(Renou, Givaudan, Poulain, Dirassouyan, & Moulin ,

2008).

2

Composition of waste in urban areas

On average, urban waste dumps consist of millions of different waste materials

(Bhada-Tata, &Hoornweg, 2012). The composition of a landfill or dumpsite gives an

idea of the physical, chemical, and thermal properties of the waste. There are four

factors that influence the composition of waste in a dumpsite: culture, seasons, laws

guiding waste disposal, and demographics of people living in the area. For example,

in developed countries and in urban areas people tend to consume more processed

foods than unprocessed food. (Bhada-Tata, & Hoornweg, 2012).

Municipal wastes are garbage collected and transported to landfills from households

and industries. Municipal waste in landfills serve as a home and substrate for

microorganisms and provide a unique ecosystem for compositing and anaerobic

digesters.

Fig.(1). Composition of household wastes in the world (Ogola, Chimuka, & Tshivhase, 2011)

3

The major challenge in the today’s world is that most of the waste generated are

inorganic, and the rate of degradation is inversely proportional to the amount of it

produced (Pettigrew, Palmisano, & Charles , 1992). In other words, the rate at which

our consumer-driven society utilizes and discards products, especially inorganic

ones, supersedes the time needed for the products to decompose. This situation is

obvious and can be found in the open garbage dumpsites in developed, developing,

and under-developed countries. In developed countries such as United States of

America, 25 million tons of plastics are discarded every year. Most of the plastics are

deposited in landfills where the degradation process may last for decades or

centuries, therefore leading to difficulties in locating new landfill sites (Pettigrew,

Palmisano, & Charles , 1992).

Open landfills are rapidly increasing due to the rate at which the world is producing

and discarding products, which has led to the extension of existing garbage sites into

new lands. New garbage sites are also being created, and this is a threat to agriculture

because it takes up land that could have been used for the cultivation of crops. An

issue with garbage dump sites is that it causes environmental pollution, such as soil

contamination and air pollution, which eventually leads to water contamination and

human health hazards such as diarrhea, respiratory ailments and dengue fever

(Agnieszka Kalwasińska, 2012).

In ancient times, when mankind relied on foraging for survival, waste management

was not a major issue because the small human population produced little waste

compared to that which we produce in tons daily (Giusti, 2009). The wastes we

produce are poorly managed globally, especially in developing countries, which is

4

causing serious environmental pollution (Giusti, 2009). The impact of improper

waste disposal can be seen in developing countries around the world.

Waste management in developing countries

Composition, waste generation, and the waste management practices in use vary

from one geographical area to another in both developed and developing countries

(Vaibhav, & Sultan, 2014). Due to the pollution caused by poor waste management

practices, there has been concern about control practices, inadequate legislation, and

the environmental and human health impacts of waste.

As a result, some countries have made efforts to enact laws that control unsustainable

Fig. (2). Waste collection data for different countries

5

waste management. However, in developing countries globally, waste may be

dumped in open landfills, without adhering to the recommended rules by the

municipal authorities. Landfills are lands where solid waste is disposed. They are

usually located at the outskirts of urban centers. Landfills are usually the final site for

waste disposal in many developing countries. This is because open-dump sites are

cheap and affordable for any nation (Vaibhav, & Sultan, 2014). However, in

developing countries, few data exist about open waste dumps, and this could be a

reason for continuous environmental pollution. . In developed countries on the other

hand, efforts have been made in reducing the amount of municipal wastes littered in

the environment. Developed countries have made modern waste management

models. These models include modern recycling, incineration, and anaerobic

digestion has been developed. In addition, countries have begun investigating a

natural form of waste clean-up: bioremediation.

Bioremediation

Bioremediation is a naturally occurring process whereby micro-organisms convert

harmful products to less toxic products (Arvanitoyannis,&Thassitou,

2001).Municipal waste acts as substrate for many microorganisms. These micro-

organisms provide a unique ecosystem for composting and anaerobic digesters. Each

layer of a landfills provide a conducive environment for microorganisms (Palmisano

& Barlaz, 1996).

The upper layer has the most nutrients because of the adequate moisture and

temperature present. A typical waste dump consists of polymeric substances, such as

paper, yard waste, and food. Therefore, waste dumps act as substrates for microbe ,

6

and these substrate will include cellulose, starch, protein, and hemicellulose. The

microbial organisms in landfills include digesters, composters, anaerobic digesters

(hydrolytic and fermentative bacteria), acetogen, methanogens and sulphate reducers.

Other micro-organisms present include such bacterial species as Bacillus spp.,

Escherichia coli, Klebsiellaspp., Proteus spp., Pseudomonasspp.,

Staphylococcusspp., and Streptococcus spp. On the other hand, fungi such as

Aspergillus, Fusarium, Mucor, Penicillium, and Saccharomyces are capable of

biodegrading waste materials (plastics). All these microbes work together to

breakdown waste as part of the bioremediation process.

Bioremediation has been found to be the most sustainable way for water and soil

remediation. This is because it does not pose a threat to the environment or human

health, and it is inexpensive (Arvanitoyannis, & Thassitou, 2001). Bioremediation

processes for the treatment of contaminated soil and water are divided into four

categories: inoculation, stimulation, use of immobilized enzymes, and use of plants.

The methods used are composting, landfarming, use of bioreactors, and intrinsic

bioremediation (Arvanitoyannis,& Thassitou, 2001).There are different types of

bioremediation that help restore polluted sites. The total number of organisms found

in a polluted area, gives an insight on how efficient the wastes can be degraded over

time. In addition, the method implore for the biodegradation also count to how fast

the waste is degraded over time. These methods includes gravimetric method and the

ohimic technology. The gravimetric method is an old method of degrading waste.

However, advanced technologies, such as the ohmic technology, have helped in

overcoming the limitations associated with characterisation of wastes for

biodegradation (Azubuike, Chikere, & Okpokwasili, 2016).

7

Bioremediation of waste materials

Biodegradation of Agricultural wastes

Keratin biodegradation

Keratin, fibrous structural protein of hair, nails, horn, hoofs, wool, feathers, and of

the epithelial cells in the outermost layers of the skin. Keratin serves important

structural and protective functions, particularly in the epithelium. It is durable

because of the cross-linkage bond between disulphide and hydrogen bonds. It is an

insoluble protein. Keratins are made up of amino acids, including cysteine, lysine,

proline, and serine(Călin, et al., 2017). However, keratin is classified into beta (hair

and wool) and alpha sheet (sheet of feathers). It can be also grouped into hard or soft

keratin (Jeffrey et al, 1995).

Keratin waste products are a threat to the environment. However, they are release in

large quantity by the agricultural industries, in the form of nails, horns, and feathers

of animals. Keratin has a high degree of stability, and can be difficult to degrade.

However, only organisms that can secrete keratinocytic enzymes, such as

keratinases, can degrade keratin (Mariana et al., 2017). Many microorganisms grow

on keratinous materials; Bacillus spp. can grow on and biodegrade keratin. The

biodegraded product can be use as feeds for chicken, fertilizers, etc (Jeffrey et al,

1995).

8

Plastic degradation

Plastics are of two types: thermoplastic and thermoset. Thermoplastics are formed by

the breaking the double bonds found in olefin (polymerization), leading to a

formation of carbon-carbon bonding. Thermosets are produced by the removal of

water from a bond between carboxylic acid and an amine or alcohol (Ying Zheng,&

Ernest K. Yanful, 2005). Plastics are polymeric molecules characterized by long

chains (Ying, Yanful, & Bassi, 2005).

Plastics are materials that can be moulded into different shapes. Although plastics are

characterized as soft, they can harden when exposed to certain factors, such as

temperature (Pettigrew, Palmisano, & Charles , 1992). After been transformed to

different shapes, plastics become stable and are difficult to degrade. Some plastics

such as Polyolefins are considered non-biodegradable because of their hydrophobic

characteristics, high molecular weight, and use of anti-oxidants (Ying, Yanful, &

Bassi, 2005).

Thermoplastic degradation

Polyolefins are thermoplastics that consist of one-carbon backbone, and they are

classified as non-biodegradable. This is because of their large molecular mass.

However, studies have shown that microorganisms can degrade thermoplastics with

low molecular mass. This is done by integration of starch such as glycerol plasticized

starch with microorganism (Keiko, Hiroshi, Yuhji , & Tani, 2001). For a plastic to

biodegradable, it must have a molecular mass of less than 500g/mole(Keiko, Hiroshi,

Yuhji , & Tani, 2001). However, the molecular mass of most plastics ranges from

40,000 to 28,000g/mole Keiko, Hiroshi, Yuhji , & Tani, 2001). For microorganisms

to biodegrade plastics, their molecular mass must be lowered. This is done modifying

9

their structure by adding a special form of synthetic Polyolefins. This structure can

then be broken down by photo-degradation, and chemical degradation leading to

short chains of carbon. Therefore, reducing the molecular mass to the acceptable

value ofless than 500 g/mol will enable biodegradation. With the molar mass of less

than 500 g/mol, microorganisms can further biodegrade polyolefins monomeric and

oligomeric: photo-degradation and chemical degradation of plastics(Keiko, Hiroshi,

Yuhji , & Tani, 2001).

Thermoset degradation

Thermoset plastics are of two types: polyurethane and polyesters. According to

Ashak,& Rathoure direct enzymatic attack on polyesters had no effect. Strains of

Trichosporum and Arthrobacter has been effective in degrading polyester (Ying

Zheng & Ernest K. Yanful, 2005). Bioremediation of polyesters by Trichosporum

and Arthrobacter occurred within one week. Polyurethanes are used in furniture and

paint. Microorganisms degrade polyurethane by attacking the urethane component.

There are three types of polyurethane bio-degradation: fungal biodegradation,

bacterial biodegradation, and degradation by polyurethanase enzymes. Examples of

fungi that perform bioremediation of polyurethane are Curvularia senegalensis,

Fusarium solani, Aureobasidium pullulans,and Cadosporium sp.. However, 100%

bioremediation of polyurethane has not been achieved (Ashak,& Rathoure, 2016).

Anaerobic digestion

Anaerobic digestion is a process whereby microorganisms break the waste in the

absence of oxygen. This process requires amicrobial community of facultative

organisms. These facultative organisms decompose waste such as food (protein,

10

carbohydrate, and lipids). The process begins with the hydrolysis of solid waste.

Solid waste is decomposed into smaller masses (monomers or dimers) by hydrolytic

organisms. These organisms can degrade soluble and non-soluble materials because

they can produce hydrolytic enzymes. The monomers or dimers are further

fermented into hydrogen, carbon, or fatty acids by acetogens and

methanogens(Maritza et al., 2008).

Other organisms involved in bioremediation of food waste are nitrate-reducing

bacteria, methanosarcina, and methanothrix. Materials that can be biodegraded by

anaerobic digester are waste food (rice, beans, yam, and so on) and paper. The

anaerobic digester method is effective. However, it becomes a problem if the

microbial organisms are unequally distributed (Alvarez, 2003). This method was

carried out in Mexico for the purpose of turning waste into organic manure in the

absence of oxygen. The major components of the digested waste were 62% paper,

23% food waste, and 15% yard clippings (Călin, et al., 2008). In this study, it was

observed that the rate of degradation is dependent on the number of organisms

present.

Bioremediation in Developing Nations

Bioremediation of contaminated soil, which includes open landfills (consisting of

keratin, plastics and food wastes), soil contaminated with oil, and water contamination

is increasingly gaining audience around the world. However, this is mostly seen in

developed countries such as United States of America. Developing countries are yet

to embrace this technology, because of the lack of technologies, and environmental

regulations that favours bioremediation. The highest rate of bioremediation comes

11

from developed nations. Although, efforts have been made by developing countries to

embrace the technology, however, however, their efforts are minimal. For example,

America (Canada and United States of America) a developed nation has implore

61.85%, of bioremediation while developing countries such as China (7.9%) Japan

(6.77%), Korea (4.5%) and India (2.93%). India as a developing nation has shown a

great zeal towards the use of bioremediation techniques ( Saraswat , 2014).

Research on bioremediation and the use of bioremediation techniques are not only

seen in Asia (developing nation), but are also seen in some African countries.

Bioremediation of contaminated soil and water have been carried out in countries

such as South Africa. In South Africa bioremediation of water contaminated with

metals was carried out in Cape Town, and isolation and characterization of engine oil

degrading indigenous microorganisms in Kwazulu-Natal (Ogola, Chimuka, &

Tshivhase, 2011). In Uganda, which is in eastern Africa bioremediation techniques

are used in cleaning up water contaminated with petroleum. In Nigeria,

bioremediation has been used in cleaning up water and land contaminated with

petroleum. However, little or no research has been conducted using bioremediation

to clean up open landfills in Nigeria as well as the whole of Africa (Ogola, &

Tshivhase, 2011).

Methods for Bioremediating waste in Developing Nations

Composting

Compositing is a bioremediation method used in remediating waste dumps in

countries such as India. Composting is the use of microorganisms to decompose

organic materials using aerobic respiration. In this process, organisms and organic

12

materials are kept under high temperature, which enables microbes to degrade waste

efficiently. This process occurs in nature, but at a slow rate, and material

decomposition is hardly noticed (Arvanitoyannis,& Thassitou2001). This

decomposition process is seen in waste dumps. To efficiently clean up waste,

microbial growth must be induced. The process starts with introducing an organism,

which is most efficient at moderate temperature of 300C to 450C (Arvanitoyannis,

&Thassitou, 2001). The organisms increase the temperature by 50C to 60C setting a

temperate for the growth of thermophile organisms, which are heat loving organisms

(Arvanitoyannis, & Thassitou, 2001). Thermophile organisms work better at higher

temperature and are continually increase the temperature around their environment.

Therefore, for maximum growth and efficient degradation to be achieved, the

temperature should be carefully monitored to avoid it browning up to 700C

(Arvanitoyannis,& Thassitou, 2001). Examples of composting microbes are

acetogens and methanogens. These organisms are used in biodegradation of plastics

(Arvanitoyannis, & Thassitou, 2001).

Bioventing

In Africa, the bioremediation process use in cleaning up soil pollutant is bioventing.

Bioventing is a bioremediation process of introducing oxygen to unsaturated polluted

site, leading to the growth of microbes. Nutrients and moistures are introduced into

the polluted soil, and this allows the growth of bioremediation organisms. The

process is more efficient when the water tables are dip within the soil structure

(Andrea, & Hinchee, 1997)

13

Fig (3): Different bioremediation techniques used across the world( Saraswat , 2014)

Case of Nigeria

It is estimated that by 2050, Nigeria will become the 3rd most populous country, and

land will be needed for agriculture and other purposes ( United Nations, 2017). Due to

poor waste management in Nigeria, open waste dumps are greatly increasing.

However, most of the waste takes years or centuries to degrade naturally, and the

finally degraded pollutants are emitted, which leads to health hazards.

However, bioremediation may be a valuable tool for developing countries such as

Nigeria for managing waste. This will lead to a healthier environment and better

human health. The efficiency of bioremediation may vary from site to site and is

dependent on the types of microorganisms present and microclimate factors, such as

humidity, soil type, and temperature. Therefore, I investigated the potential of

microorganisms found in open dumpsites in Jimeta-Yola in north-eastern of Nigeria

to perform bioremediation was investigated. The study was aimed to determine what

14

microorganisms are present in the soil in the selected site and how well they perform

bioremediation on solid waste.

15

AIMS & OJBECTIVES

Aims

To investigate the ability of microorganisms to perform bioremediation on solid

waste from dump sites in Jimeta-Yola, north-eastern Nigeria

Objectives

• To collect soil samples from different segments of the waste dump in Jimeta -

Yola

• To culture the isolated microorganisms.

• To identify the types of microorganisms found in the solid waste dump site.

• To isolate microorganisms with solid waste materials.

• To evaluate degradation of waste over time by each microorganism.

16

CHAPTER 2

MATERIALS & METHODS

Study Site

In Yola, improper waste management is prevalent. Thus, we conducted this study in

Yola, the state capital of Adamawa State, Northeastern Nigeria. The waste dump is located

at the outskirt of Yola, and it is located along a commercial road. There are farm lands and

a stream just beside the waste dump. The most cultivated around the dump site are

vegetables. Other commercial activities such as mechanic workshops are located just a

meter from the dump site. The open landfill has an oozing smell.

Inorganic waste products can be found littered around all over the dump. The

improper disposer of waste in Nigeria is due lack of law enforcement on citizens.

The constituents of the waste heap burnt include household wastes such as clothing

materials, paper, and spoilt food among others. These municipal wastes pose two

Fig (4): Map of Yola town.

17

threats to the soil quality in the area. Leachate occurs when it rains, and wastes are

increasing rapidly due to high population.

Sampling

Source of Soil samples were collected from the dumpsite described above. In order

to obtain the soil sample, the surface debris was removed, and the subsurface soil

dug to a depth of 30 cm with a hand shovel. Soil samples were dug from depths of 0-

30cm and transferred to a sterile container within the perimeter of each refuse

collection. One thousand five hundred grams (1500 g) of soil from dumpsites were

collected in three different sterilized containers, at three different locations. All

samples were labeled sample A-E and the temperature of each sample (50oC) was

determined immediately after collection at each waste collection point using a

thermometer before they were transported to the laboratory for analysis immediately

after collection.

Isolation of the microorganisms

The soil samples were sieved through a 0.2 mm wire mesh, which was previously

treated with 95% ethanol, to obtain fine soil particles (U.S. EPA, 1978)..Isolation of

the bacteria was carried out by weighing 1 g of the soil sample aseptically into 10 ml

of distilled water in a conical flask. .The suspension was mechanically shaken for 10

min at room temperature. One milliliter (1 ml) of the suspension was diluted with 9

ml distilled water. This dilution was repeated four times in series resulting in a 10-4

final dilution. Aliquots (100 µl) of each appropriately diluted sample were inoculated

using spread plate method onto 15plates of minimal media such as rubber and agar,

agar and water, (LBA) ,and Trypticase soy agar (TSA) that were prepared and

18

autoclaved at a temperature of 1210C. The culture plates were incubated at 30 ºCfor7

days. After incubation, the plates were observed for bacteria growths. The plates

were further purified by sub-culturing three to four times to obtain pure bacteria

isolates. The pure cultures obtained were spread on LB/ Rubber media, to see

whether they can degrade plastic. The isolate bacteria grown on plastics, and LB

media were inoculated in a liquid broth to test whether they can actually degrade

Polypropylene.

Microscopy and Biochemical Identification

The 24 hrs old pure culture was used in preparing smear by placing an aliquot of the

bacteria culture on the slide containing a drop of water and was heat fixed. A smear

of bacteria was applied on to a slide. Air dried and heat fix by passing it through a

flame 5 times. Five (5) drops of Hucker’s Crystal Violet was added to the culture,

and was allowed to stand for one minute. The slide was washed with water, and

excess water was shaken off. Thereafter, 5 drops of iodine solution were added to the

culture, and allowed to stand for 30 seconds. The slide was washed briefly with

water, tilted, decolorized with alcohol and washed immediately with water. Five (5)

drops of Safranine O were added to the slide, and allowed to stand for 1min. The

prepared slide was examined under microscope at both 400 x and 100 x.

Different biochemical test such as citrate test, starch hydrolysis, indole, methyl red,

VP, glucose, lactose, and arabinose test were carried out by weighing appropriate

grams and dissolved with distilled water and autoclaved at 121oC for 15 min per

square inch. The 24 hr old culture of the isolates were inoculated to each of the test

samples and incubated at 37 oC for 24 - 48hr. The result of each test was recorded.

19

Catalase and oxidase test were also carried out by dropping the hydrogen peroxide

and oxidase kovac reagent on a slide followed by addition of an aliquot of the

bacteria isolate and within 3 sec the results were observed

Carbon content of biodegraded samples using Fourier Transform Infra red (FTIR)

The isolated organisms were inoculated in the rubber broth that was incubated in a

shaker for two weeks. The incubated broth was shaken, and 2 ml of the culture was

poured in the test tube using a pasture pipette. About 2 ml of dichloromethane was

added to the test tube. The mixture separated into three different layers when

shaken for 15 min at a speed 85000 rev/min. The mixture was removed from the

centrifuge and a drop of the lower layer was applied on the plate surface, which was

left to dry for 30 sec. The carbon content was determined using the infrared

spectrophotometer.

The transmittance values obtained from the IR results were used to calculate the

carbonyl index (Equations 2.1 and 2.2)

(2.1)

where A is absorbance and T is the transmittance. Equation 2.1 was used to

determine the absorbance values.

(2.2)

TA %-2=

1464

17801700=

A

Adexcarbonylin

20

CHAPTER 3

RESULTS

In this study, we obtained six different strains of bacterial in 3 grams of collected soil

samples. Three of the strains were capable of performing biodegradation (Fig 6). One

of the strains was capable of performing biodegradation. The Actinomycyte

(biodegradable bacteria) was morphologically different when cultured on a different

media. Actinomycyte took a minimum of two days for a visible growth to occur.

When the temperature increased from 370C to 400C, a rapid growth of asynomycyte

was observed on a rubber media and it was green in color. Some of the microbe

found in the soil grew on a minimal media instead of on a rich media such as LB

agar. The inoculated bacteria utilized carbonyl residues and reduced its

concentration. The IR results show that the control group had more carbon groups

than the experimental group (those with bacteria). The carbonyl index results

determined from the transmittance percentage of the carbonyl peaks (Equation 3.1)

was in this following order: degraded sample 1(1.16) > degraded sample A5 (0.94) >

degraded sample E (0.85).

21

22

Table 1:.IR interpretation chat for all sample after 2 week of biodegradation

B A C D

Pea

k

(cm-

1)

Functiona

l groups

Peaks(c

m-1)

Function

al groups

Peaks(c

m-1)

Function

al groups

Peaks(c

m-1)

Function

al groups

690 C-H

vibration

1371 CHOH 1372 -CH3 1375 -CH3

100

0

C-O 1458 CH2 1456 CH2 1459 CH2

121

6

C-H 1589 C-C

stretchin

g

1469 CH3 bend 1718 C=O

(Ketone)

137

0

-CH3 2846 C-H 1719 C=O

(Ketone)

2850 C-H

142

5

C-O

vibration

1739 C=O

(ester)

2856 CH2 2925 C-H

stretchin

g

159

1

C-C

stretching

2924 C-H

stretchin

g

174

2

C=O

(aldehyde

)

2926 C-H

stretchin

g

287

5

-CH2 3625 OH

292

6

-CH2

asymmetr

y

3750 OH

337

5

OH

364

4

OH

376

2

OH

23

Table 2: Biochemical and morphological test for all organism gotten from the waste

dump

Test Actinomycyte

spp

Organism

A

Organism

1

Organism

2

Organism

3

Organism

4

Catalase - - + - _ +

Oxidase + + - + + _

Indole - - - - _ _

Citrate - - - - _ +

MVRP - - - + _ _

Gram

staining

+ + + + + +

Fig (6). Color change in broth as a result presence of Psuedomonas spp

24

The experimental sample was cloudier and milky compared to the control group (Fig

6) when shaken; however, when undisturbed, the experimental sample had a green

color.

25

CHAPTER 4

DISCUSSION

This study showed that the microbial community found in waste dumps are capable of

degrading polypropylene. Although, the microbial analysis results (Table 3.2) pointed

towards the presence of Actinomycyte in the soil sample, the colour changes observed

in the samples could be an indication of other bacteria such as Pseudomonas spp. The

presence of other bacteria growth in the rubber media could be as a result of

interspecific completion leading to death of Actinomycyte or it could be as a result of

co-metabolism.

Actinomycyte spp are gram-positive organisms, which are generally

anaerobic bacteria. Actinomycyte spp are different from other gram-positive

organisms due to their filamentous and branching growth pattern that results, in most

forms, in an extensive colony, or mycelium. Many species of Actinomycyte occur

in the soil and are harmless to animals and higher plants. However, some are

important pathogens and many others are beneficial sources of antibiotics and the

degradation of plastic. However, the species obtained during the research is notable

for its ability to secrete antibiotics and its ability to degrade polypropylene.

Morphologically, Actinomycyte spp was observed to grow in a rod-like manner,

sticking firmly to the growth media (Mogoşanu, 2016).

The presence of the functional group C-C(at peaks 3625 and 3750 cm-1) in sample A

and B was not surprising as a different method was used for sample A (co-

metabolized), which lead to the similarities between samples A and B. The presence

of ketone and ester functional groups in the biodegraded samples confirmed that

26

degradation actually took place because of microbial activities. When polymers such

as PP are exposed to microorganisms capable of degrading the polymers, new

functional groups such as alcohol, ester, and ketones are formed (Klein, 2016).

As earlier stated, the presence of green color in the biodegraded sample could be due

to the presence of other bacteria such as pseudomonas spp. The production of water-

soluble diffusible pigments is an indicator use to identify Pseudomonas species. The

bluish- green pigmentation is a sole indicator for identifying pseudomonas spp

(Alberto, 1981).

Morphologically, pseudomonas spp was observed to grow in a rod-like manner. It is

capable of growing on either a minimal media or rich one. In addition, it can also

grow on rubber. Other studies have shown that most robust growths of pseudomonas

are on carbon rich media. Pseudomonas is a facultative organism that when supplied

with nitrogen grows on plastic (Aamer, 2008). Therefore, getting nitrogen from the

air and carbon atom from polypropylene, Pseudomonas spp is capable of degrading

polypropylene. The nitrogen from air was as a result of opening the prepared culture

the closed chamber (Aamer, 2008).

The structure of polypropylene used for the biodegradation study comprises of a

hydrogen atom linked to a tertiary carbon atom in the backbone. This property makes

polypropylene degrade preferentially by chain scissor method. This leads to a change

in molecular weight during the distribution curve. In addition, microorganisms

usually release enzymes capable of metabolizing one substance into another.

27

Polypropylene has three majors C-C, C-H and a methyl functional group. However,

during the experiment, three other functional groups appeared. These were ester,

ketone and aldehyde. In addition, microbial activities led to degradation of the

polymer. The IR results showed that there was an absence of peak at 2830 – 2700

which occurred in sample C and D and CH3 and C-H functional groups. These were

seen at 1370 an 1216 cm-1. The band at 1739 indicates ester, 690, 1000, 1216 cm-

1bands of C-H, C-O and C-H, respectively are not in the degraded samples. Also,

3375, 3644 and 3762 cm-1 bands were present in control (sample B), but absent in

degraded samples C and D. The degraded sample A had OH bands at 3625 and 3750

cm-1 and C-C bands were only present in the control sample B and degraded sample

A (Klein, 2016).

.

Esters are chemical compounds derived from carboxylic acids. Carboxylic acids

contains a COOH group and the H bond is replaced with a methyl, ethyl and

sometimes a phenyl group.

28

Both ketone, ester and aldehyde belong to a carbonyl functional group (carbon-

oxygen double bonds). The difference between aldehyde and ketone is that the

carbonyl group bonds to at least one hydrogen atom. In ketone, the carbonyl group

bonds to two carbon atoms.

A peak at 2398 represents either an isocyanides functional group or a C=C. This is

because both functional groups have the same peak range (2000-2500), which makes

it difficult for one to differentiate them except with the use of advanced devices such

as GCSM or ultraviolet spectrometer machine. These machines are capable of

identifying every functional group, irrespective of the similarities in the peak height.

However, if the peak at 2398 is a cyanide group, this implies that the bacteria is a

nitrogen-fixing one and that it can convert atmospheric ammonia to nitrogen. In

addition, bacteria comprise of proteins and they secrete enzymes that contain protein.

Fig. (10). Molecular structures of aldehyde and ketone

Fig.(9) Molecular structure of ester

29

The nitrogen in the protein reacts with carbon to form an isocyanide functional group

at peak 2398 (Klein, 2016).

The C=C can also be found at the peak range of 2000-2500. The presence of C=C

implies cracking of polypropylene by Actinomycyte spp. The hydrogen carbon bond

is broken and electrons transfer to the next carbon. The functional group changes

from alkane (saturated hydrocarbon) to alkyl (unsaturated hydrocarbon) making it

easier to break the bonds. This implies the biodegradation of the material, which is

polypropylene (Klein, 2016).

The presence of a C-C bond implies an interspecific competition between the

organism that leads to the ability of the bacteria present in the soil to convert C-C

bonds to either carbonyl or ester functional group (Klein, 2016).

For catalase test, the negative sign (-) indicates no color change while the positive

sign (+) represents a color change. The oxidase test indicates the presence of bubbles

when a scoop of the organism interacts with hydrogen peroxide.

The positive sign (+) in gram staining test indicates a color change (purple) on the

organism’s cell membrane.

Starch agar is a differential medium that tests the ability of an organism to produce

certain extracellular enzyme such as a-amylase and oligo-1,6-glucosidase. This helps

to hydrolyse starch. This happens because polymers such as starch are

macromolecules that are unabsorbed directly into the bacteria cells except when

broken down into subunits. Microorganisms that are able to degrade starch produce

30

extracellular enzyme that degrades starch. An organism hydrolyses starch when there

is absence of colour after the addition of iodine. A clearing around the bacterial growth

indicates that the organism has hydrolysed starch. On the other hand, iodine turns blue,

purple, or black (depending on the concentration of iodine) in the presence of starch.

This indicates that the starch is not hydrolysed. In Table 4.2, all organisms cultured

during the experiment are capable of digesting starch (Hopkins, 1954).

The indole test is used for testing the ability of certain bacteria to decompose the amino

acid tryptophane to indole. Tryptophan is an amino acid that can undergo deamination

(the removal of an amino acid from a substance or a compound), and it is hydrolysed

by bacteria capable of producing tryptophanase. Indole is generated by

reductive deamination from tryptophan via the intermediate molecule indole pyruvic

acid. Tryptophanase catalyzes the deamination reaction during which the amine (NH2

group) of the tryptophan molecule is removed. This means that organism B is capable

of secreting tryptophanase enzyme. Organisms that possess tryptophanase can carry

out the following reaction: l-tryptophan → indole + pyruvic acid + NH3.

Citrate agar is used to test an organism’s ability to utilize citrate as a source of

energy. The medium contains citrate as the sole carbon source and inorganic

ammonium salts (NH4H2PO4) as the sole source of nitrogen. Bacteria that can grow on

this medium produce an enzyme, citrate-permease and is capable of

converting citrate to pyruvate. Positive reaction indicates microbial growth with

colour change from green to intense blue along the slant, while negative reaction

indicates no microbial growth alongside with no colour change, that is, slant remains

green. Only one organism (organism 4) found in the waste dump tested positive

31

(capability of using citrate as its carbon source, and ammonium salt). Organism 4 is

capable of using any carbon source and nitrogen from the atmosphere to make energy

for its survivor.

This test demonstrates the presence of catalase, an enzyme that catalyses the release

of oxygen from hydrogen peroxide (H2O2). It is used to differentiate bacteria that

produces an enzyme catalase from a non-catalase producing bacteria. The enzyme

catalase mediates the breakdown of hydrogen peroxide into oxygen and water. The

presence of the enzyme in a bacterial isolate is evident when a small inoculum is

introduced into hydrogen peroxide and the rapid emergence of oxygen bubbles occurs.

The lack of catalase is evident by a lack of, or weak bubble production. Only organism

2 and 4 tested negative for catalase test.

Hydrogen peroxide is found in our bodies, and it affects our hair colour change during

ageing. They are three types of growth: anagens, catagens, and tolagens. During the

anagen, the accumulated hydrogen peroxide sits in the papillary cavity where it

bleaches the hair as it grows – no different than if we bleached your hair yourself with

a bottle of bleach. Over time it is also possible for the melanocyte cells to die off

because the hydrogen peroxide interacts with other crucial enzymes such as MSRA

and MSRB and deactivates them. This prevents the hair follicles from repairing itself

leading to other aspects of aging hair such as thinning and weak/brittle hairs. This can

result in a whole lot of grey hair. With advanced research on organism 2 and 4 grey

hair that occur during the anagen due to accumulation of hydrogen peroxide can be

avoided.

32

Gram staining is a common technique used to differentiate groups of bacteria based

on the constituents of their cell wall. The Gram stain technique distinguishes between

Gram positive and Gram negative groups. One can differentiate the two groups base

on colour staining of the cell wall, which is either purple (positive) or red/pink

(negative). Gram positive bacteria stain violet because of the presence of thick layer

of peptidoglycan. Peptidoglycan is an essential component of the bacterial cell wall,

and it protects the cell from bursting due to turgor and maintains cell shape. Composed

of glycan chains connected by short peptides. peptidoglycan forms a net-like

macromolecule around the cytoplasmic membrane. It helps protect bacterial cells from

environmental stress and helps preserve cell morphology throughout their life cycle

(Hsu, & Nieuwenhze, 2015). All bacteria screened during the experiment are gram

positive. This implies that they will better adapt to stress and prevent its cell from

bursting (Hsu, & Nieuwenhze, 2015).

33

CHAPTER 5

CONCLUSTION

Waste generation will be in existence as far as the human being lives, and waste

production will continue to grow as human population increases. In most developing

countries, theses wastes are improperly disposed leading to environmental

degradation. However, bioremediation has been tested against all odds as the

cheapest and environmentally friendly method for the degradation of waste.

However, most developing countries are not aware of this cost efficient method.

Developed countries have developed tactics to clean up their environment.

Nevertheless, there is hope for Nigeria and other developing countries.

Bioremediation is the hope, and effective waste managing scheme of Nigeria lies in

bioremediation.

Recommendations

I was unable to identify other biodegradable species. In addition, this study may

have benefitted more from the use of sophisticated machines, and a longer duration.

A longer duration and provision of these machines appears important for

identification of organisms capable of performing bioremediation, and visible or

greater changes in degradation of materials.

34

REFERENCES

Saraswat, (2014). A review of waste management practices and their impact on

human health Waste Management. Journal of Bioremediation and

Biodegradation. Retrieved from www.omicsonline.org/open-access/patent-

analysis-on-bioremediation-of-environmental-pollutants-2155-

6199.1000251.pdf

Alvarez, M. J. (2003). Bioremediation of Mnucipal Solid Waste. (M. J. Alvarez, Ed.)

London: IWA.

Andrea, & Hinchee. (1997). Soil bioventing principles and practice. washington

D.C: Lewis Publisher. Retrieved from

books.google.com.ng/books?hl=en&lr=&id=zAujoFw86ScC&oi=fnd&pg=P

R5&dq=bioventing&ots=EHX7hZKBvn&sig=hVFWXnIDGkNcBSQG3N_k

3NVPXUA&redir_esc=y#v=onepage&q=bioventing&f=false

Anna Palmisano C and Morton Barlaz A. (n.d.). Microbiology of Solid Waste. 1996:

CRC Press.

Arvanitoyannis,Thassitou & I.S. (2001). Bioremediation: a novel approach to food

management. Trends in Food Science & Technology, 12 , 185–196.

Ashak & Rathoure. (2016). Toxicity and waste management using bioremediation.

(R. V. Ashak K., Ed.) IGI.

Ashak K., R. (2016). Toxicity and waste management using bioremediation. (R. V.

Ashak K., Ed.) IGI.

Azubuike, Chikere, & Okpokwasili. (2016). Bioremediation techniques–

classification based on site of application: principles, advantages, limitations

and prospects. World Journal of Microbiology & Biotechnology, 32, 80.

35

Bhada-Tata, &l Hoornweg. (2012). What a Waste: A Global Review of Solid Waste.

Word Bank.

Bhada-Tata, D. H. (2012). What a Waste: A Global Review of Solid Waste. Word

Bank.

Bhattacharyya, T. K. (2012). Municipal Solid Waste Generation, Composition, and

Management: The World Scenario. Environmental Science and technology,

42:15(1064-3389), 1509-1630.

Ghosh, Adhikari, & Ray, as cited in Ying Zheng & Ernest K. Yanful. (2005). A

Review of Plastic Waste Biodegradation. A Critical Review in Biotechnology,

25 , 243–250. doi:10.1080/07388550500346359

Giusti, L. (2009). A review of waste management practices and their impact on

human health. Waste Management, 29, 2227–2239.

Goran B. Boskovic, Mladen M. JosiJevic Neboj M. Jovicic & Milan J.Babic. (2016).

Co-generation potentials of municipal solid waste landfills in Serbia. 20(4),

pp. 1271-1281. doi:10.2298/TSCI150626063B

Keiko, Hiroshi, Yuhji , & Tani. (2001, May). Degradation of polyethylene by a

fungus, Penicillium simplicissimum YK. Polymer Degradation and Stability,

72(2), 323-327.

Kerazun z.m, Indeka L.G as cited in Agnieszka Kalwasińska. (2012). Sanitary

Quality of Soil in and near Municipal Waste Landfills Site. Pol. J. Environ.

Stud, 21(6), 1651-1657.

Liyan Song, S. Y. (2017). Geographic and environmental sources of variation in

bacterial. Appied Microbioogyl Biotechnoogyl, 101, 761-769.

doi:10.1007/s00253-016-7917-6

36

Mariana et al. (2017). Degradation of keratin substrates by keratinolytic fungi.

Electronic Journal of Biotechnology, 28, 101-112.

Maritza et al. (2008). Anaerobic digestion of municipal solid waste and agricultural

waste and the effect of co-digestion with dairy cow manure. 99, pp. ) 8288–

8293.

Muhammad Rafiq, Huma Salma Giliani,Munir Khan,Muhammad Atiq. (2015).

Health and economic implication of solid waste dumpsites: A case study

Hazar Khwani Peshawar. FWU Journal of Social Science, 9(2), 40-52.

Muhammad, Huma, Munir, & Atiq. (2015). Health and economic implication of

solid waste dumpsites: A case study Hazar Khwani Peshawar. FWU Journal

of Social Science, 9(2), 40-52.

Nations, U. (2017). Nigeria to become 3rd most populous country by 2050. Retrieved

from www.premiumtimesng.com/news/top-news/234712-nigeria-become-

3rd-populous-country-2050-un-report.html

Ogola, Chimuka, & Tshivhase. (2011). Management of Municipal Solid Wastes: A

Case Study in Limpopo Province, South Africa. Retrieved from

www.intechopen.com/books/integrated-waste-managementvolume-

i/management-of-municipal-solid-wastes-a-case-study-in-limpopo-province-

south-africa

Palmisano & Barlaz . (n.d.). Microbiology of Solid Waste. 1996: CRC Press.

Pettigrew, A. C. (1992, October). Biodegradability of Plastics. American Institute of

Biological Sciences, 2(9), 680-685.

Pettigrew, Palmisano, & Charles . (1992, October). Biodegradability of Plastics.

American Institute of Biological Sciences, 2(9), 680-685.

37

Renou, Givaudan, Poulain, Dirassouyan, & Moulin . (2008). Landfill leachate

treatment: Review and opportunity. Journal of Hazardous Materials, 150,

468–493.

Renous S, G. J. (n.d.). Landfill leachate treatment: Review and opportunity.

S. Renou, J. G. (2008). Landfill leachate treatment: Review and opportunity. Journal

of Hazardous Materials , 150, 468–493.

S. Renou, J.G. Givaudan, S. Poulain a, F. Dirassouyan, P. Moulin c,. (2008). Landfill

leachate treatment: Review and opportunity. Journal of Hazardous Materials,

150, 468–493.

Silva & Alvarez. (2010). Bioaugmentation. In K. N. Timmis (Ed.), Handbook of

Hydrocarbon and Lipid Microbiology (pp. 4531-4544). Springer.

T. Mandri and J. Lin. (n.d.). Isolation and characterization of engine oil

degradingindigenous microrganisms in Kwazulu-Natal, South Africa. African

Journal of Biotechnology, 023-027.

T.C Hazan. (2010). Biostimulation. In K. N. Timmis (Ed.), Handbook of

Hydrocarbon and Lipids Microbiology. Springer.

Vaibhav Srivastava, S. A. (2014). Urban solid waste management in the developing

world. Rev Environ Sci Biotechnol. doi:10.1007/s11157-014-9352-4

Vaibhav, & Sultan. (2014). Urban solid waste management in the developing world.

Rev Environ Sci Biotechnol. doi:10.1007/s11157-014-9352-4

Ying, Yanful, & Bassi. (2005). A Review of Plastic Waste Biodegradation. Critical

Reviews in Biotechnology, 25, 243–250.

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