Ee Gung Bohun
Ee Gung Bohun
Ee Gung Bohun
BY
SUBMITTED TO
AUGUST, 2024
Certification
EEGUNGBOHUN JANET Blessing with matric number 2211920060 and it has been read
and approved as meeting the requirements for the award of Higher National
Diploma (HND) in the Microbiology, faculty of Pure and Applied Sciences, Osun
____________________________ _______________
____________________________ __________________
MR AGBESANYA
All praises, adoration and glorification due to Almighty God the most beneficent, the most
merciful, the ancient of days, the creator and sustainer of the universe for sparing my life till this
My highest gratitude goes to my beloved, caring, amazing and wonderful parents for their love,
care, special advice and support rendered enormously throughout my Higher National Diploma
(HND).I pray to Almighty God to continue to shower his endless blessing and mercies upon you
and spare your life to reap the sweet fruit of your labour in sound health, long life, prosperity,
I acknowledge my supervisor Miss Omole who has nature in spite of their extremely busy and
tight schedule to read through my project report and her guidance through the success of the
project .I say's thank you ma. I will not forget to thank my benefactor of knowledge, my able
H.O.D. Mr Agbesanya for his fatherly roles and words of encouragement and to all other viable
lecturer's in the department of microbiology, who has contributed in one way or the other and
positively in planting me quality education and moral of high value. I say's a big thanks to you
A dumpsite is an arena specifically used for the disposal of wastes. It is an old traditional
method of waste disposal similar to landfill method of waste management. The aim of this study
is to isolate, characterize and identify bacteria present in different dumpsite in Esa Oke town.
Isolation was done by serially diluting the soil samples and pour plated on nutrient agar,
MacConkey agar, and EMB agar, then incubated at 37oC for 24 hours. Colonies developed after
24 hours were subcultured and stored for biochemical characterization. The organisms isolated
in this study include Proteus vulgaris, Proteus mirabilis, K.pneumonia, Citrotrobacter ferundii,
Citrobacter diversus, Providentia retgerri. The presence of these potential pathogens reported in
the present investigation may be attributed to the disposal of raw human faecal discharges and
other human wastes at the waste-dump site. The health hazard associated with the indiscriminate
dumping of waste around residential areas and other ecologically sensitive areas such as rivers
and streams and arable land cannot therefore be under-estimated. Nigeria should therefore
direct her efforts towards the treatment of waste before disposal as to minimize the health
CONTENTS PAGES
Title page i
Certification ii
Dedication iii
Acknowledgement iv
Abstract v
Table of contents
CHAPTER ONE
1.1 Introduction 1
CHAPTER TWO
CHAPTER FOUR
CHAPTER FIVE
5.1 Summary 34
5.2 Conclusion 34
5.3 Recommendation 34
References
CHAPTER ONE
Introduction
Soil is generally a favourable home for the increasing the number of microorganisms, with micro
colonies, developing around soil particles. Number of microorganisms in soil habitats normally
are much higher than those in fresh water or marine habitats (Atals & Bartha, 2008).
Bacteria and fungi actively participate in organic matter decomposition liberating chemical
nutrients and increase plant growth. Microorganism numbers vary in and between different soil
types and conditions, with bacteria makes up the most abundant in soils. Bacteria counts in
different soil ranged from 4x106 to 2x109 dry soil (Whitman et al., 1998). Grayston et al., (1998)
reported that the growth of microbial population and their action on soils are dependent on the
interaction between plant species and soil. Marschner et al., (2001), reported that bacterial
community composition results from the interaction between soil type, plant species and its
rhizosphere localization. According to Wieland et al., (2001); Pinto & Nahas, (2002), the size
and structure of microbial population are affected by soil type and plant species.
Soil microorganisms have been isolated and quantified using several selective and non-selective
culture media (Sorheim et al., 2009) that have some advantages over non culturable methods,
allowing for taxonomic, genetic and functional studied in the isolated microorganism. According
to Kennedy & Gewin, (2007), different growth rates are often seen in microbial population from
different soils, which have different nutrient requisites that are not supplied by the media.
Moreso , many media containing different nutritional requirements have been suggested for the
estimation of bacterial diversity (Balestra % Misaghi, 2007), which include saline media
prepared using a chemically defined mixture of salts and a carbon source, as well as a complex
media, rich in polypeptides, amino acids and vitamins and mineral (Seeley et al., 2001).
Microorganisms are known to be ubiquitous in nature (i.e. can be found everywhere) and have a
whole lot of advantages and disadvantages (disease causing organisms), therefore it is imperative
to analyze soils from dumpsite to be able to known the type of microorganism that can be found
there and possible danger the organisms can pose on human when we get in contact with soil
from dumpsites.
The main aim of this study is to isolate organisms from soil of dumpsites and characterize the
organism isolated to be able to know their identity and their possible effect to human.
Objectives
This study will provide information of the type of organisms inhabiting different dumpsites.
This study is limited to isolation of bacteria and fungi, other group of microorganisms such as
CHAPTER TWO
Literature Review
2.1 Soil
Soil is normally considered as the fine earth which covers land surfaces as a result of the in situ
weathering of rock materials or the accumulation of mineral matter transported by water, wind,
or ice (Sharma, 2019). The distinctive feature of soil is that to this weathered mineral material is
added organic material. This organic material may be both living and dead. The dead organic
matter will include little altered and freshly added dead plant roots and leaf and other plant litter,
dead fauna, and organic material in various stages of decomposition from little modified
relatively fresh materials to the complex decomposed material called humus. It is this mixture of
mineral and organic material which gives the soils their distinctive characteristics (Rasool,
2018). Across the surface of the earth there are many different types of soil which reflect, at least
in part, varying combinations of mineral and organic matter and their differing responses–both
separately and often in complex association–to different environmental conditions. Indeed soil
(and the soil constituents), together with the plant life it supports, the rock on which it lies, and
Depending upon the context, the word ‘‘soil’’ may have very different meanings. A simple
definition of soil is the material that plants grow in and which provides them with physical
support, water, and nutrients. There are other more particular uses of the term soil. To the
engineer, soil is the finely divided and relatively loose ‘‘rock’’ material at the earth’s surface,
and this overburden is often considered an inconvenience because it must be removed. The
geologist calls this layer the regolith and geological investigations frequently begin below it. The
hydrologist looks on the soil as if it were a large reservoir storing water to supply streams and
rivers. The most widely held view of the soil, however, is as a medium for plant growth and the
provision of food and fiber directly or through the intermediate stage of animals (Araujo, 2018).
Soil is important as a medium for plant growth and for the support of much animal and human
activity (Abarkeli, 2016). The soil acts as a reservoir for nutrients and water providing the plants’
needs for these requirements throughout their growth. The soil may also provide an environment
for the breakdown and immobilization of materials added to the surface (in addition to the
aforementioned plant and animal remains) such as fertilizers and pesticides and waste products
such as sewage sludge, animal wastes and slurries, and composted refuse materials (Monterio,
2018). The soil is a complex dynamic system in which the interactions of the biological,
rendering initially harmful materials less dangerous and immobilizing others as a result of the
interactions between these added materials and the organic and inorganic soil constituents. This
immobilization may enable breakdown of the potentially dangerous materials to less dangerous
forms. These interactions and transformations may be long term, over decades, medium term
over months or years, short term between individual events such as rainstorms, or almost
that country. The total mass of the microflora and -fauna beneath the soil is 20 times that of the
human population of the earth (Torsvik et al. 2019). One gram of healthy soil contains 1 million
to 100 million bacteria involved in the breakdown of organic matter, 0.15–0.5 mg of fungal
help retain soil particles as macroaggregates/clumps, while the remaining components of the soil
food web maintain the correct number of bacteria and fungi by prey–predator interactions, and
thus help in the recycling and retention of basic nutrients in the soil. This is necessary to
These components of the soil food web exist in harmony and are intricately woven in
interdependent relationships (Johansson et al. 2016). The structure and function, i.e. number,
activity and community structure of the soil food web, may act as a prime indicator of ecosystem
health. Monitoring the active and total biomass of each organism group directly may help in
2.3 Fungi
Fungi are living organisms that are distantly related to plant, and are more closely related to
animals, but rather different from either of those groups. Fungi are eukaryotic that, like algae,
have rigid cell walls and may be either unicellular or multicellular. Some may be microscopic in
size, while other form much larger structure, such as mushrooms and bracket fungi that grow in
soil or on damp logs. Unlike algae, fungi do not contain chlorophyll and thus cannot carryout
photosynthesis. Fungi do not ingest food but must absorb dissolved nutrients from the
environment of the fungi classified as microorganisms, these that are multicellular and produce
filamentous, microscopic structures are frequently called molds, whereas yeasts are unicellular
In molds, cell are cylindrical in shape attached end to end to form thread like filaments (hyphae)
that may bear spores. Individually, hyphae are microscopic in size. However when large numbers
of hyphae accumulate for example, on a slice bread or fruit jelly, they from a fussy mass called a
mycelium that is visible to the naked eye. The unicellular yeasts have forms, from spherical to
egg-shape to filamentous. Yeasts are noted for their ability to ferment carbohydrates, producing
alcohol and carbon dioxide in product such as wine and bread (Sneath et al., 2006).
Fungi are large organisms that usually live on dead and rotting animal and plant matter. They are
found mostly in soil, on objects contaminated with soil, plants and animal, and on skin and they
may also be airbone. Fungi may exist as yeasts or molds and may alternate between the two
(0.0001 to 0.0002 inch) in diameter. Molds consists of filamentous branching structures (called
hyphae) , 2 to 10 miconietres in diameter, that are formed of several cells lying end to end
Fungi can produce their mycotoxins under laboratory conditions or naturally in various
agricultural products (Massey, et al., 1995). Fungi cause a significant yield reduction and
economic losses because it's commonly contaminate food and crops. It addition, they changes the
appearance, taste, texture and order to food, and also unsafe for human consumption because of
their mycotoxins. The consumption of foods which contaminated with mycotoxins are associated
with several cases of human poisoning, sometimes resulting in death. (Molina and Gianuzzi.
2009). Nowadays, mycotoxins have been receiving worldwide attention and several group of
mycotoxins are known as ergot, afflatoxins, ochratoxins, citrinin, patulin and fumonisines
(Mohanamba et al., 2002). Fusarium, aspergillus and penicillium were noted to be the major
fungal populations in feeds and foods. F. monilifome present in the feed and food for about a
year. The predominant naturally occurring fungi belonging mainly to penicellum purpurogenum,
Importance of fungi: fungi inhabit almost every niche in the environment and humans are
2 Biosynthetic factories. The fermentation property is used for the industrial production of
10 Fungi (leptolegnia caudate and Aphanomyceslaevis) are used to trap mosquito larves in
paddy fields and thus help in malaria control (Saleemi et al.. 2010)
2.6 Soil Pollution
Soil pollution as part of land degradation is caused by the presence of xenobiotics chemicals or
other alteration in the natural soil environment (Davies, 2005). It is typically caused by industrial
activity, agricultural chemicals or improper disposal of waste. The most common chemicals
lead, and other heavy metals (Scott, 2012). Contamination is correlated with the degree of
industrialization and intensity of chemical substance. The concern over soil contamination stems
primarily from health risks, from direct contact with the contaminated soil, vapors from the
contaminants, or from secondary contamination of water supplies within and underlying the soil
(Rao et al., 2017). Mapping of contaminated soil sites and the resulting cleanups are time-
consuming and expensive tasks, requiring extensive amounts of geology, hydrology, chemistry
and computer modeling skills as well as an appreciation of the history of industrial chemistry
(Yang et al., 2021). Developing countries tend to be less tightly regulated despite some of them
having undergone significant industrialization. It is important to understand that all soils contain
compounds that are harmful/toxic to human beings and other living organisms. However, the
concentration of such substances in unpolluted soil is low enough that they do not pose any
threat to the surrounding ecosystem. When the concentration of one or more such toxic
substances is high enough to cause damage to living organisms, the soil is said to be
3.1 Materials
Test tubes
Conical flasks
Beakers
Measuring cylinder
Inoculating loops
Spatula
Weighing balance
Bunsen burner
Incubator
Autoclave
Staining rack
Microscope
Paper tape
Sterile syringes
Forceps
Methylated spirit
Hand glove
Sterile nylon
Permanent marker.
Nutrient agar
Oil immersion
All glassware carefully washed with detergent and rinsed with tap water followed by distilled
water. They were then set to drain out and before sterilization in the hot air oven at a temperature
3.2.2 Autoclaving
Culture media and distilled water were sterilized in the autoclave at 121 °C for 15 minutes.
3.2.3 Flaming
Inoculating loops were sterilized using Bunsen flame. The mouth of conical flask were flamed
before media were poured into petri dishes, inoculation were done near bunsen burner flame to
avoid contamination.
Five different sample of dumpsite soil were collected from different point in Esa Oke, samples
One gram of soil from each of the five samples was weighed and added aseptically into sterile
test tubes which contain 9ml of sterile distilled water each and shaken thoroughly for even
distribution of organisms to make a stock. Five-fold dilution was carried out by transferring 1ml
each of the mixture into the sterile test tubes that contain 9ml of sterile distilled water labeled to
make 10mL using a new sterile syringe 1 ml of the solution that is already been mixed and
shaked was dispensed from test tube labeled into another test tube labeled 10' 2 containing 9ml of
Distinct colonies were then sub-cultured by streaking on a fresh solidified agar and incubated
appropriately. A pure culture of each colony type on each plate was obtained and maintained.
Microorganisms
3.4.1 Macroscopy
Microscopy examination of the fungal plates were done with the naked eye on day-to-day basis,
from first day of inoculation for a period of a week for features such as formation of colonies, the
texture, size and colour of the colonies from both the front and reverse sides of the saboraud
3.4.2 Microscopy
The microscopic observation of the fungi isolates was performed with the use of a microscope.
The technique of James & Natalie (2011) was adopted for identification of the unknown isolated
fungi using lactophenol cotton blue stain. Young cultures (5 days old) were used so as to avoid
overgrowth of the organisms that may lead to difficulty in viewing distinct features under the
microscope.
The identification was achieved by placing a drop of the stain on clean slide with the aid of a
mounting needle a small portion of the mycelium from the fungal cultures was removed
aseptically and placed on the drop of lactophenol cotton blue on the glass slide. The mycelium
was spread uniformly on the slide with the needle. A cover slip was gently applied on the slide
with little pressure to eliminate air bubbles. The slide was then mounted and observed under x l0
Gram staining
Microscopic morphology of the isolates was studied using the Gram staining technique
(Harrigan & McCance, 1998). A thin smear of 18-24 h old culture of each of the isolate was
made on clean grease free slide and heat-fixed by passing over a bunsen burner flame. Two
drops of crystal violet was added to the smear and then allowed to stay for 60 seconds. The
crystal violet was washed off with distilled water and then flooded with Gram’s iodine for 60
seconds. The Grams iodine was washed off with 95% ethyl alcohol for 15 seconds. Two drops
of safranin solution was then used to counter stain the smear and allowed to stay for 30 seconds
before rinsing off with distilled water. The slides were then allowed to air dry after which they
were observed under oil immersion objective lens of a microscope. Gram positive bacteria were
stained purple, while Gram negative bacteria stained red. The shape and arrangement of cells of
Catalase test was carried out using the slide method. A colony of 18-24 h old culture of the test
isolate was mixed with a drop of freshly prepared 3% hydrogen peroxide solution on a clean
grease free microscope slide. The occurrence of effervescence indicated gas evolution
representing a positive catalase reaction, while the absence of gas evolution indicated a negative
H2O2→2H2O + O2 (g)
Tryptone broth (1% w/v) was prepared as directed by manufacturer and 10 mL was dispensed
into each McCartney bottle and then sterilized by autoclaving. Inoculation of the bottles was
carried out by using 48 hours old culture of the isolates. The isolates tubes and a control tube
were incubated in anaerobic jar for 48 hours. After incubation, a few drops of Kovac’s reagent
was added to tryptone basal culture medium. Formation of red or pink ring at the top which is
concentrated by alcoholic layer was taken as positive indole test while negative indole test is
indicated by the primary yellow colour of the medium and the control. The test confirms the
ability of the test organisms to produce indole from amino acid trytophan (Oyeleke et al., 2006).
(c) Oxidase test
A what man filter paper (No 1) was soaked with oxidase reagent (1% aqueous tetramethyl-p-
phenylenediamine hydrogen chloride) and placed in a sterile petri-dish. A sterile wire-loop was
used to pick 24 hours old culture of the organism from the plate and used to touch the filter paper
containing oxidase reagent. The development of purple coloration within 5-10 seconds signified
an oxidase positive reaction while a delayed reaction or no colour change indicates a negative
reaction (Olutiola et al., 1991). The test confirms whether there is presence or absence of certain
oxides in the bacteria that will catalyse the transport of electrons between electron donor in
About 5 ml of sterile MRVP medium (Glucose phosphate broth) in test tubes was inoculated
with 0.1 ml of 18-24 h old of the isolates and incubated at 35℃ for 5 days (Harrigan &
McCance, 1998). The MRVP culture (1.0 mL) was transferred to a clean test tube and few drops
of methyl red indicator was added. A resultant red coloration was indicative of a positive
The ability of the microorganisms isolated to produce acetoin was tested as described by
Harrigan & McCance (1998). Sterile five millilitres (5 mL) of MRVP broth in test tubes was
inoculated with 0.1 mL of 18-24 h old MRS broth culture of the test isolates with an
uninoculated broth serving as control. The inoculated medium was incubated at 35° C for 5 days
and 1 mL of the MRVP culture was tested by adding 0.5mL of 6% α-naphthol and 0.5 mL of
16% KOH. The test tube was shaken thoroughly and left to stand. Appearance of a red coloration
within five minutes was indicative of the production of acetoin which constituted a positive
result.
CHAPTER FOUR
4.1 Results
Table 4.1: The mean of the total viable counts of the isolates in cfu/ml from dumpsite soil
1 DS1 2.8
2 DS2 1.28
3 DS3 1.72
4 DS4 2.0
.
Table 4.2: Morphological Characteristics of the Isolated Bacteria
DS1b Weak+ + _ _ _ + + + _ _ + _ +
DS2 + Weak + + _ + + + + _ _ _ +
a. Discussion
The bacteria isolated included Proteus vulgaricus, Proteus mirabilis, Klebsiella pneumonia,
Citrobacter ferundii, Citrobacter divrsus, and Providentia retgerri. The isolation of these from
the dump sites is an indication that microbes are not only ubiquitous in nature but also populate
the soil, thus increase the nutritional value of the soil. These microbes produce enzymes which
All the bacterial isolates reported in this study have been reported to be associated with waste
and waste biodegradation. Faecal coliforms and Providentia retgerri have been reported to be
associated with waste (Ekundayo, 2007). Klebsiella, and Pproteus were also reported by Cook et
al., (2004). The present investigation has revealed the presence of various bacteria known to be
associated with waste biodegradation and their frequency of isolation. The activities of these
bacteria if properly harnessed can be used in future treatment plants in Nigeria in accelerating the
bioconversion of waste compost into organic fertilizer for use in gardening, agriculture and
horticulture.
All the bacterial genera reported in this study have been reported by Cook et al (2004) and
Monica Chesborough (2005) as potential pathogens. That is, they are capable of causing disease.
The presence of these potential pathogens reported in the present investigation may be attributed
to the disposal of raw human faecal discharges and other human wastes at the waste-dump site.
CHAPTER FIVE
CONCLUSION AND RECOMMENDATION
5.1 Conclusion
The isolation, characterization, and identification of microorganisms from dumping site soil
provide valuable insights into the environmental impact and potential risks associated with
lain/soil pollution. The findings help in implementing necessary safety measures, controlling
5.1 Recommendation
The recommendations are being proposed for consideration, further research could be conducted
to compare the performance or efficiency of organism that can bio remediate soils in dumping
References
Ameen, I.A., Danjuma, B.Y., & Raji, O.Y. (2018). Isolation and Identification of Hydrogen
Barnett, L., & Hunter, B. (2018). Illustrated general of imperfection fungi (3rd ed.) USA: Burges
Publishing Company.
Bidoia, C., Adenipekun, O., Oyetungi, O. S., & Kassim, C. Q. (2017). Biodegradation of used
Motor Oil in Soil Using Organic Waste. Biotechnology Research International 6(3): 66.
Blodgett, A. (2018). Growth response of Ricinus communis L. (Castor Oil) in spent lubricating
oil polluted soil. Journal of Applied Science and Environmental Management 9 (2): 73-79.
Challian, A. Ugoh, S. S. and Moneke, L. U. (2004). Isolation of bacteria from engine oil
Cheesbrough, T. (2005). Hydrocarbon-degrading filamentous fungi isolated from flare pit soils
phytochelatins in heavy metal stress tolerance of plants. South African Journal of Botany
76: 167-179.
Ekhaise, N. & Nkwelle, A. (2018). Isolation of bacteria from mechanic workshops’ soil
(22): 6301-6303.
Folasade, A. (2015). Metal concentration in plant tissues of Ricinus communis L. (castor oil)
grown in soil contaminated with spent lubricating oil. Journal of Applied Science and
Fawole, M.O., & Oso, B.A. (2004). Laboratory Manual of Microbiology. Spectrum Books
George, U., Tasie F., & Muotoe F. (2019). Hydrocarbon degradation potentials of indigenous
fungal isolates from petroleum contaminated soils. Journal of Physical and Applied
Sciences. 3:1-6.
Jai, S.P., Tiwari, K.L. & Jadhav, S.K. (2015). Long Term Preservation of Commercial Important
Khan, J.A., & Rizvi, S.H.A. (2018). Isolation and characterization of microorganisms from oil
Lloyd, N.R. (2018). Lubricating oil-degrading bacteria in soils from filling stations and auto-
Lloyd, E. & Cackette, A. (2017). Biodegradation of used engine oil by fungi isolated from
mechanic workshop soils in Sokoto metropolis, Nigeria. Sky Journal of Soil Science and
Megharaj, S.J., Singleton, I., McClure, N.C. & Naidu, R. (2017). Influence of petroleum
445.
Nwoko, C. O., Okeke, P. N., Agwu, O. O., & Akpan, I. E. (2015). Performance of Phaseolus
vulgaris L. in a soil contaminated with spent engine oil. African Journal of Biotechnology
6(16): 1922-1925.
3(5): 483-489.
Olukunle, O.F., Boboye, B. (2016) Phylogenetic Analysis of Oil Degrading Bacteria Associated
with Polluted sites in river State, Nigeria. Arch. Applied Science Research, 4(4):1600
Oyeleke S.B., & Manga S. B. (2018). Essentials of microbiology practical to best publishers 33-
60.
Okon, A. (2016). Bailey and Scott’s diagnostic microbiology. 13 th edition. St. Louis, Missouri:
Elsevier.
Omotayo, A.E., Ojo, O.Y., & Amund, O.O. (2012). Crude Oil Degradation by Microorganisms
Olukunle, O.F., & Boboye, O. H. (2013). Effects of petroleum spills on the germination and
growth of Glycine max (L.) Merr. Nigerian Journal of Botany. 16: 76-80.
Rao, A. Ying, Y., Sheng, T. & Xue, B. (2017). (2018). Effect of spent engine oil on growth
446- 450.
Raven, A. Obire, O., Anyanwu, E.C. & Okigbo, R.N. (2005). Saprophytic and crude oil-
degrading fungi from cow dung and poultry droppings as bioremediating agents.
Rolling, R., Wang, J. J., Jiq, C. R., Wong, C. K. & Wong, P. K. (2012). Characterization of
olyclic aromatic hydrocarbons created in lubricating oils. Water, Air and Soil pollution.
120: 381-396.
Scott, A. (2012). Impact of heavy metals pollution on soil organic matter accumulation. Journals