Research proposal

Aggreh Erhovwon Peter Page 1
EXECUTIVE SUMMARY
INTRODUCTION
The study will involve the use of organic (cow dung (C), cow dung slow release (CDSR)) and
inorganic fertilizers (urea direct release (UDR), urea slow release (USR)) as well as the
combinations of the both types of fertilizers in the biostimulation of oleophilic microbes in
an ex-situ bioremediation of a polluted soil sample collected from Bodo community, Gokana
Local Government Area of Rivers state, Nigeria. This project reveals the greatest secrets of
time as it affects the removal of Total Petroleum Hydrocarbon (TPH) in a shell polluted site
as a result of pipeline disruptions activities as well the enumeration of the relative
abundance and diversity of the oleophilic microbes. A soil physicochemical analysis of the
polluted site was carried out to determine the presence of the various bioremediation
factors such as electrical conductivity, nutrients, pH, soil moisture, oxygen content, and low
clay or silt content, aerobic or anaerobic that is required for a microbial remediation of a
polluted site oleophilics microbe in a contaminated crude oil sample will be stimulated with
the various nutrient composition combinations. The biodegradation process will be
monitored by monitoring THB, HUB, PAHs, TPH and Nitrate during the process. A soil
physicochemical analysis of the polluted sample will be first carried out wherein various
parameters will be analyzed. A baseline result of physicochemical properties of the polluted
sample will thereafter be obtained. A proximate analysis on the organic fertilizers as well as
the methods of preparations will also be highlighted during the process. Lastly, purification
and identification of hydrocarbon utilizing bacteria will be determined to ascertain the
relative diversity and abundance of the oleophilic microbes and a statistical graphical
description or presentation of the abundance and diversity of the oleophylic microbes
isolated will be obtained and analyzed.
Keywords: Oleophylics, bioremediation, THB, HUB, physicochemical tests. Etc.

INTRODUCTION
Microbial communities can be extremely diverse. This is especially the case for soil microbial
communities. (Delmont et al, 2011). The traditional culture method or techniques in
determining the abundance and diversity of oleophilic microbes is highly specific though
cannot be compared to the molecular pattern because of its limitations.
Bioremediation is not a panacea but rather a natural process alternative to such methods as
incineration, catalytic destruction, the use of adsorbents and the physical removal and
subsequent destruction of pollutants. Many industrialize activities as seen in the
manufacturing companies have resulted in vast pollution to our environment. This has
immediate and long term effects on the environments. It is pertinent to note that the
quality of life is always linked to the overall quality of the environment.
Due to the numerous advantages of bioremediation ranging from cost to safety,
bioremediation is a paradigm for a fast growing population. Bioremediation uses biological
agents, mainly microorganism’s i.e. fungi, yeast or bacteria to clean up contaminated soil
and water (Strong and Burgess, 2008).

LITERATURE REVIEW
AN OVERVIEW OF REMIDIATION
Bioremediation can be defined as any process that uses microorganism or their enzymes to
return the environment altered by Contamination to its original condition. (Kumar A, et al
.,2011). It is a process whereby organic wastes are biologically degraded under controlled
conditions to an innocuous state or to levels below concentration limits established by
regulatory authorities. (Mueller 1996). Biodegradation of a compound is usually as a result
of multiple oleophilic microbes.
The rate of childbirth increases day by day in an uncontrolled manner especially in the
underdeveloped & developing counties as compared to the death rate. This dramatically
increases the population statistics. As the population increases, more industries are been
built to meet up the demands of the increasing population, and hence more pollution to the
environment. Man’s life depends on the safety of the environment, for this reason and
many other reasons it is pertinent to look for a approach that can not only help in pollution
control but whose after effects in terms of production of bio surfactants is not adverse to
the environment. Bioremediation provides solution to this puzzle as compared to other
evolutionary pollution control methods.

TYPES OF BIOREMEDIATION
Generally bioremediation can be classified into two broad categories.
1. In situ bioremediation
2. Ex situ bioremediation
In situ bioremediation is usually done at the point of contamination. The contaminant is not
excavated. It is further divided into intrinsic and engineered bioremediation while situ
bioremediation requires the removal of the contaminant from the point of contamination.
It is divided into the solid phase system and slurry phase system of ex situ bioremediation
which are further divided into various systems as explained in the table below.
A TABLE SHOWING VAIOUS BIOREMEDIATION TECHNIQUES.
Technique Examples Benefits Application
In Situ Biosparging
Bioventing
Bio
augmentation
Most efficient,
noninvasive.
Relative passive
Naturally attenuated
process, treat soil and
water
Biodegradation abilities of
indigenous microorganisms.
Presence of metals and
inorganic compounds.
Environmental parameters,
biodegradability of pollutants.
Chemical solubility, geological
factors, distribution of
pollutants.

Bio
stimulation
Rapid increase in
biodegradation
Nutrients and electron
acceptors stimulate activity of
microorganism.
Ex situ Land farming
(solid-phase
treatment
system)
Composting
(Anaerobic,
converts solid
organic
wastes into
humus –like
material)
Biopiles
Cost efficient, simple,
inexpensive, self-
heating.
Low cost and rapid
reaction rate,
inexpensive, self-
heating.
Can be done on site
Surface application, aerobic
process, application of organic
materials to natural soils
followed by irrigation and
tilling.
To make plants healthier, good
alternative to land filling or
incinerating practical and
convenient.
Surface application, agricultural
to municipal waste.
Biorectors Slurry
reactors
Acqueous
reactors
Rapid degradation,
kinetic optimized,
environmental
parameters.
Enhances mass
transfer, effective use
of inoculants and
surfactant.
Toxicity of amendments
Toxic concentrations of
contaminants
Precipitation Non- directed Cost effective Removal of heavy Metals

or flocculation physic-
chemical
complex
reaction
between
dissolved
contaminants
and charged
cellular
components
Microfiltration Microfiltration
membranes
are used at a
constant
pressure
Remove dissolved
solids rapidly
Waste water treatment;
recovery and reuse of more
than 90% of original waste
water
Electro
dialysis
Uses cation
and anion
exchange
membrane
pairs
Withstand high
temperature and can
be reused.
Removal of dissolved solids
efficiently
Source: Google downloads

ADVANTAGES OF BIOREMEDIATION
1. It is generally recognized as being less costly than other remedial options, (e.g pump
and treat, excavation).
2. Can be combined with other technologies (e.g., bioventing, soil vapour extraction) to
enhance size remediation.
3. In many cases, the process dues need produce waste products that must be disposed
of due to adverse effect on environment.
4. Bioremediation can be carried out on site without the disruption of every day to day
activity.
5. It can destroy a wide variety of contaminants.
6. It is generally acceptable by the public due to the fact that it is a natural process.
7. It can be carried out without the transfer of contaminants from one site to the other.
DISADVANTAGES OF BIOREMEDIATION
1. Injection wells and/or infiltration galleries may become plugged by microbial growth
or mineral precipitation.
2. High concentration (TPH>50,000 ppm) of low solubility constitutes may be toxic
and/or bioavailable.
3. Difficult to implement in low-permeability aquifers (<10-4 cm/sec)
4. Re-injection wells or infiltration galleries may require permits or may be prohibited
5. Some states require permit for air injection.
6. May require continuous monitoring and maintenance
7. Remediation may only occur in more permeable layer or channels within the aquifer.

REQUIREMENTS FOR MICROBIAL GROWTH IN BIOREMEDIATION
PROCESS
Requirement Description
Carbon
source
Carbon is the most basic element of living forms and is needed in greater
quantities than other elements. Carbon contained in many organic
contaminants may serve as a carbon source for cell growth. If the organism
involved is an autotroph CO2 or HCO3 in solution is required. In some cases,
contaminant levels may be too low to supply adequate levels of cell carbon,
or the contaminant is metabolized via co-metabolism. In these cases the
addition of carbon sources may be required.
Nutrients The growth and activity of the microorganisms must be estimated by
adequate maintenance and supply of nutrients. Bio-stimulation usually
involves the addition of nutrients and oxygen to help indigenous
microorganisms. These nutrients are the basic building blocks of life and
allow microbes to crea
te the necessary enzymes to break down the contaminants. Nutrients:
Nitrogen (ammonic, nitrate, or organic nitrogen) and phosphorous (ortho-
phosphate or organic phosphorous) are generally the limiting nutrients. In
certain anaerobic systems, the availability of trace metals (e.g. iron, nickel,
cobalt, molybdenum and zinc) can be of concern.
Source: From Stainer et al. (1986), Microbial World, 5th Ed., Prentice Hall,
NJ.
Energy
source
In the case of primary metabolism, the organic contaminant supplies energy
required for growth. This is not the case when the contaminant is
metabolized via secondary metabolism or co-metabolism or as a terminal
electron acceptor. If the contaminant does not serve as a source of energy,
the addition of a primary substrate(s) is required.
Electron All respiring bacteria require a terminal electron acceptor. In some cases,

acceptor the organic contaminant may serve in this capacity. Dissolves oxygen is a
common electron acceptor in aerobic bioremediation processes. Under
anaerobic conditions, NO3-, SO43-, Fe3+, and CO2 may serve as terminal
electron acceptors. Certain co-metabolic transformations are carried out by
fermentative and other anaerobic organisms, in which terminal electron
acceptors are not required.
Temperature Rates of growth and metabolic activity are strongly influenced by
temperature. Surface soils are particularly prone to wide fluctuations in
temperature. Generally, mesophilic conditions are best suited for most
applications (with composting being a notable exception).
pH A pH is another important factor that influences bioremediation process. If
the soil is acidic, it is possible to raise pH by adding lime. A pH ranging
between 6.5 and 7.5 is generally considered optimal. The pH of most ground
water (8.0–8.5) is not considered inhibitory.
Source: B Pandey et al. (2012)
Other requirements includes: absence of toxic metals, soil moisture, adequate contact between
microorganisms and substrates requirements, time and environmental requirements.

MICROORGANISMS NECESSARY FOR BIOREMEDIATION PROCESS
The first organisms registered with the ability for hydrocarbon degradation was Pseudomonas putida
in 1974 (Prescott et al, 2002). These microorganisms can be classified either as aerobic, anaerobic,
fungi E.tc.
These microorganisms includes; Acinethobacter, Actinobacter, Acaligenes, Arthrobacter, Bacillus,
Berigerinckia, flavobacterium, Methylosinus, Mycobacterium, Mycococcus, Nitrosomonas, Norcardia,
Penicillum, Planerochaete, Psudomonas, Rhizoctoma, Seratia, Xanthofacter (B Pandey et al, 2012)
PRINCIPLES OF BIOPREMEDIATION
Bioremediation of a compound is often a result of the actions of multiple organisms. For
bioremediation to be effective, microorganisms must enzymatically attack the pollutants and convert
them to harmless products and converts them to harmless products microorganisms feeds on the
hydrocarbon thereby decreasing the quantity and they also produces bio surfactants which emulsify
these pollutants into harmless substances. (Vidali 2001).
BIOCHEMICAL TESTS
These are tests carried out on a bacteria isolate to determine the identification and characterization
of the isolate. It is usually compared to a Berger’s manual of determination bacteriology/
The tests includes IMViC, MRVP oxidase D Xylose, L-Rhamnuse, Tricholosem Endospores, Gram
Staining, Morphology, microscopy, macroscopic, Growth at 41oc and 4oc, colony characteristics, etc.

HOW ARE PETROLEUM COMPOUNDS FORMED?
Petroleum is a term derived from a branch of chemistry known as organic chemistry. Organic
chemistry is the study of carbon compounds. It means that it is not only the study of compounds from
nature but also synthetic compound.
TPH refers to the measurable amount of petroleum based hydrocarbons in an environmental matrix.
Thus while PHC deals with an absolute and somewhat intangible quantity; TPH pertains to actual
results obtained by sampling & analysis. (Ross Sadler et al, 2015)
It is also important to point out that, during a bioremediation process, the increase of the HUB is
directly proportional to the decrease of the total petroleum hydrocarbon.
TPH is one of the physicochemical parameters that is to be determined before a bioremediation
process.
HOW DO HYDROCARBONS GETS INTO THE ENVIRONMENT (SOIL & WATER)
There are different routes for the entry of hydrocarbon to the environment. The most common of this
is leakage from underground storage tanks. Other major sources include spillage during refuelling &
lubrication. Places whereby there transfer & handling of crude oil also potential places of
contamination, ( Rose Sadler, et al. 2015)
In Nigeria, hydrocarbon rapidly gets into the soil as a result of illegal pipeline disruptions thereby
causing serious leakage.

CHEMICAL COMPOSITION OF SPILLED PETROLUEM HYDROCARBON
Petroleum hydrocarbon molecules are mainly saturates aromatics, resins and asphaltenes. (Ma
Caulay BM et al, 2014).
The degradation of those components by microorganisms is as follows; Alkanes>Light
aromatics>Cycloalkanes>Heavy aromatics> Asphalthenes. (Hamne et al, (24)). Resins degrade
naturally because of the little complexity.
The following table lists the properties of a range of alkane’s fractions that can be found at
contaminated site.
SIMPLE PARAFFIN ALKANES
Molecular
Formula
Name Boiling Point
(o C)
Melting Point
(o C)
Density at
20o C
C6H14 n-Hexane 69 -94 0.658
C8H18 n-Octane 126 -98 0.702
C10H22 n-Decane 174 -32 0.747
C12H26 n-Dodecane 215 -12 0.768
C16H34 n-Hexadecane 287.5 18 0.775 (at mp)
C20H42 n-Eicosane 205 36.7 0.778 (at mp)
C30H62 n-Triacontane 449.7 66 0.775
C35H72 n-Pentatriacontane 490 74.6 0.781

The table below summarizes biodegradability of some contaminants.
HYDROCARBON CONTAMINANTS AND THEIR LEVEL OF DEGRADATION.
Contaminants Level
Simple hydrocarbons, C1-C15 Very easy
Alcohols, Phenols, Amines Very easy
Acids, esters, amides Very easy
Hydrocarbons, C12-C20 Moderately easy
Ethers, monochlorinated hydrocarbons Moderately easy
Hydrocarbons greater than C20 Moderately difficult
Multichlorinated hydrocarbon Moderately difficult
PAHs, PCBs, pesticides Very difficult
Source. Google downloads.

STATEMENT OF PROBLEM
The effect of direct application of inorganic fertilizers to the environment when used as a bio
stimulant during a bioremediation process.
AIM
The aim of this experimental work is to provide a systematic understanding of the various processes,
advantages, disadvantages and mechanisms involve in a laboratory bioremediation process when
using a slow release NPK fertilizer (srNf), NPK direct release (Ndr), cow dung fertilizer (cdf) as well as
the determination of the relative abundance and diversity of the oleophilic microbes involved in the
process.
OBJECTIVES
The objective of this experimental work is to provide a systematic understanding of the various
process & mechanisms involve in a laboratory bioremediation process when using a slow release NPK,
urea and an inorganic droppings & Cow dung fertilizers & the determination of the abundance &
diversity of the oleophilic microbes involved in the process.
SPECIFIC OBJECTIVES
To determine and establish the baseline properties of the soil sample using microbiological and
physiochemical analysis.
To determine the effects of various single and in combination of organic and inorganic direct and slow
release fertilizers in the bio stimulation of oleophilic microbes in polluted sample.
To determine the diversity and abundance of oleophilic bacteria involved in a bioremediation process.
To ascertain whether the slow release fertilizer will be good for stimulating hydrocarbon utilizing
bacteria.

MATERIALS AND METHODS
DETERMINATION OF BASELINE PROPERTIES OF THE SAMPLES
The following soil physicochemical properties parameters will be analysed during the exercise. They
are as follows; pH, electric conductivity, nitrate, phosphate, Total Organic Carbon (TOC), heavy metals
(zink, nikel and lead), PAHs and temperatue.
pH
The pH of the polluted sample will be determined using a pH conductivity meter (Jenway 3015
model). 50ml of distilled water will be added to 5.0g of the soil sample. The lump of the soil will be
stored to form homogenous slurry then the probe of the pH meter will be immersed into the sample
and allowed to stabilize at 25oc and the pH of the sample will be determined. (ISSAH 2013).
NITRATE
The nitrogen content of the polluted sample will be determined using a brucine reagent. One millilitre
of the soil filtrate will be measured into a clean sterile test tube and 1ml of distlled water will be
measured into another test tube to serve as a blank solution. Half millilitre of brucine reagent will be
gently introduced into both test tubes. Two millilitre of concentrated sulphuric acid will be then be
added and shaken to homogenize. The resulting solution will be allowed to cool to room
temperature. The solution will then turn yellow and will be measured at 470nm on a
spectrophotometer to determine the nitogen content in the soil sample. (Frank 2012).
PHOSPHATE
2 grams of the soil sample will be weighed and placed in a silica crucible and ashed at 550oc in a muffle
furnace for 4 hours. The ash residue will be dissolved in a 4ml dilute HNO3 filter paper in a 50ml
volumetric flask and the volume will be carried out in the dry ashed sample solution with the aid of a
specthrophotometer. (ISSAH 2011).
TOTAL ORGANIC CARBON (TOC)
The wet oxidation technique will be used. One gram of sample will be transferred into a clean a clean
pyrex conical flask. Five millilitres will be heated on an electro thermal heater for 15min to reflux. The
sample will then be cooled to room temperature and diluted to 100ml with distilled water. Twenty
five millilitre of the sample solution will be treated with 0.2M ferrous ammonium sulphate using

ferrion as indicator. A blank containing oxidant (potassium chromate) and sulphuric acid will then be
titrated as in the sample and the titre value will be recorded. Calculation will be made using the
formulae below to get the TOC value. (Frank et al,. 2012)
%TOC = Titre value of blank –sample titre × 0.003 × 100
Sample weight
POTASSIUM
Two grams of the soil sample will be weighed and placed in a silica crucible and ashed at 550oc in a
muffle furnace for 4 hours. The ash residue will be dissolved in a 4ml dilute HNO3 filtered through an
acid washed paper in a 50ml volumetric flask and the volume will be made up to the mark. The
estimate of potassium concentration will be carried out in the dry ashed sample solution with the aid
of a spectrophotometer.
DETERMINATION OF ELECTRICAL CONDUCTIVITY (EC)
The soil sample will be collected in a sterile polythene bag making sure that contamination of the
sample is avoided. The bag will be air dried for a few hours thereafter mix in the bag to ensure a
homogenous sample and then with the aid of a sieve probably with approximate 2mm spacing,
sample will be sieved to remove any large soil clumps. ½ of a cup of the dried soil will be measured
and put into a glass beaker. ½ of a cup of distilled water will be added to the glass beaker. The
mixture will be stirred gently for 30 seconds. N.B. the mixture will not be mix too harshly in order not
to destroy the humus structure which determines its elements. The soil water suspension will be left
to stand for 30 minutes. The water will then be stir gently again and the EC measurement will
thereafter be taken. The EC meter will be inserted into the beaker and it will be swirl gently around
the soil water extract. After approximately 30-60 seconds or after the EC reading has stabilized, the
EC reading will be read and displayed on the EC meter. (www.agriculturesolutions.com).
TEMPERATURE
The pots will be incubated in the open but under a shade at approximately 28 to 30oc which later will
be determined by the use of a thermometer. The mean daily temperatures of the soil blend will be
taken in the morning (6am), afternoon (12 noon) and evening (6pm). The mean values will then be
calculated.

SOIL TEXTURE TYPE (STT)
HEAVY METALS (ZINK, NIKEL and LEAD)
MOISTURE CONTENT DETERMINATION
TOTAL PETROLEUM HYDROCARBON (TPH)
The extraction of petroleum hydrocarbon will be done with dichloromethane (DCM) using cold
extraction method with ASTM D-3694 heavy machine for 1 hour. Procedurally, 20g of dried soil
samples will be weighed into 100ml conical flask. Twenty grams of activated anhydrous sodium
sulphate and 20ml of DCM will be gently added into the barrier containing the test sample. This will
be allowed to stand for 1 hour and then filtered into 50ml conical flask using filtration plugged/
packed with cotton wool. Procedure is repeated on the residual soil until a colourless solution will be
obtained. The extracts will be analysed by Gas chromatography using HP Agilant 6890 gas
chromatography equipped with a FID detector, an Agilant 7673 auto sampler and 5 capillary column
(15m×0.25mm) with a nominal film thickness of 0.25µm split less injection method (all in batch).
Injection volume will be 1µl and injection temperature of 330oc. Helium will be used as a carrier gas
(2ml/mm). The column will be held at 35oc for 150 minutes. Real values of TPH will be calculated as
products of raw data on FID table on graph and dilution factor used for the sample. (Frank et al.,
2012).
ENUMERATION OF TOTAL HETEROTROPHIC BACTERIA (THB)
THB counts will be determined using spread plate count agar (PCA). From each sample, 1g or 1ml will
be homogenized in 9ml of 0.85% normal saline using Heindoph Vortexing machine. Decimal dilutions
(10 fold) of the suspensions will be plated out on agar medium and incubated at 30oc for 24 hours.
The colony forming units will be afterwards enumerated. (Chioma and Chioma, 2014).
ENUMERATION OF HYDROCARBON UTILIZING BACTERIA (HUB)
Hydrocarbon utilizing bacteria (HUB) will be enumerated by a method adopted from Chioma and
Chioma, (2014) which will involve the dilutions of the sample and plating out on a mineral salt
medium (Bushnell-Hass Agar) (Sigma-Aldrich, USA). Hydrocarbon will then be supplied to putative
hydrocarbon utilizes by placing sterile Whatman No. 1 filter paper impregnated with 5ml crude oil on
the lids of the inverted plates and incubated for 14days at 30oc. Colony forming unit (Cfu/g) will
thereafter be calculated.

DETERMINATION OF TOTAL HYDROCARBON CONTENT (THC)
The THC will be analysed using the standard solvent extraction method. A gram of the sieved soil
sample will be dissolved in chloroform in a test tube. Thereafter, the clear layer will be collected with
a clean test tube upon which it will be dehydrated by the addition of a spoonful of anhydrous sodium
sulphate. The clear extracted solution will then be absorbed at 420nm HACH DR/2010
spectrophotometer. The THC concentration will be extrapolated with a reference from a standard
curve obtained from the graph of produced crude oil at varying concentrations.
PREPARATION OF ORGANIC MANURE
COW DUNG
Cow dung of about 3kg will be obtained from cow slaughter house in mile 3 market Rivers state. The
cow dung will be sun dried for three weeks until moisture is driven out completely. The cow dung will
then be grinded into powdered form. The grinded cow dung will be passed through a 2mm standard
mesh sieve thereafter some samples of the powdered cow dung will be sent for determination of its
mineral content.
FORMULATION OF SLOW RELEASE FERTILIZER
UREA
5g each of coconut coir dust, cassava mesocap and wheat will be added to 10kg solid granular urea
fertilizer. This will be mixed homogenously to obtain a slow release urea fertilizer.
PROXIMATE ANNALYSIS
COW DUNG:
GOAT DROPPINGS:
COCONUT COIR DUST:
CASSAVA MESOCAP:
WHEAT:
PURIFICATION AND IDENTIFICATION OF HYDROCABON UTILIZING BACTERIA
Distinct colonies of the HUB will be randomly picked using a sterile wire inoculating loop and sub
cultured for purification by streaking on nutrient agar plates which will be incubated at 30oc for 24

hours. Individual colonies with distinct colony characterization that shows ability to utilize
hydrocarbon in the Bushnell hass agar medium will be examined macroscopically, microscopically and
also identified using biochemical tests as described in Bergy’s manual for determination of
bacteriology. (Chioma and Chioma, 2012).
STUDY AREA
The hydrocarbon polluted soil was obtained from a shell polluted soil in organic land in Rivers State,
Nigeria. There are various roads that connect to this site. This area was selected because of the high
rate of pollution.
SAMPLE COLLECTION
Using a spade, the sample was collected in a plastic bucket. Several points in the contaminated site
were sampled to enhance accuracy.
BIOREMEDIATION SET UP
Four kilograms each of the polluted soil sample placed in five different 5 litres pots with 1cm
diameter openings at the base. The pots will be in triplicates to represent five different treatment
regimens namely Urea direct release (Udr), Urea slow release (Usr), Urea slow release + Cow dung
slow release (Usr+Cdsr), Cow dung direct release (Cddr) and lastly Control (C). The oil contaminated
soil samples will be thoroughly mixed with a hand trowel sanitized with 70% ethanol before applying
the various regimens in the respective soil sample setup. Microcosms will be kept at room
temperature. Nutrient treated soils will be regularly watered weekly with 200ml sterile distilled water
to substitute for evaporated water and will also be mixed every day for aeration purposes. The
microcosms (i.e the various set up’s) will be sampled at days 0, 7, 14 and 35. Triplicate microcosms
will be thereafter be sampled and the following parameters will be monitored during the experiment;
TPH, HUB, PAHs, THB, THC, Nitrate and Phosphate.
EXPERIMENTAL GROUP TEST EXPERIMENT
EXPERIMENTAL GROUP TEST EXPERIMENT

SET A 4kg of polluted (P) soil + 2kg of Udr
SET B 4kg of polluted soil + 2kg of Usr
SET C 4kg of polluted soil + 2kg of (Usr + Cdsr)
SET D 4kg of polluted soil + 2kg of Cddr
SET E (control) 4kg of polluted soil only.
SCHEMATIC REPRESENTATION SHOWING POTS IN TRIPLICATES
SET A 4kg P soil + 2kg Udr
SET B 4kg P soil + 2Kg Usr
SET C 4kg P soil + 2kg (Usr+Cdsr)
SET D 4k
SET E
SETUP A
0th day 7th day 14th day 28th day
Urea Slow
Release (S/R) +
Cow dung
Urea S/R + Goat
droppings
Control

SETUP B
NPK Slow Release
(S/R) + Cow dung
NPK S/R + Goat
droppings
Control
SETUP C
NPK + Cow dung
NPK + Goat
droppings
Control

SETUP D
Urea + Cow dung
Urea + Goat
droppings
Control
PHYSICOCHEMICAL ANALYSIS
PH
The pH of the sample was determined using a pH meter.
CONDUCTIVITY
The conductivity of the sample was determined using a conductivity meter values µs/cm.
MEASUREMENT OF NITRATE
The brucine method was used in the nitrogen determination.
MEASUREMENT OF PHOSPHATE

The calorimetric method as described by the United Nations Environment Programme
(UNGP, 2004) was used.
TOTAL ORGANIC CARBON
The wet oxidation techniques previously reported by (Nelson & Sommers, 1975) was used.
Other parameters tested were; moisture, potassium, magnesium, sodium, aluminum,
hydrogen, base saturation, total petroleum hydrocarbon, soil texture type.
DETERMINATION OF TOTAL HETEROTROPHIC BACTERIA (THB)
Total heterotrophic bacteria count present in the triplicates in the various groups will be
determined at the day 0, 7, 14, & 28 day of the experiment. To do this, the plate count agar
was used for the culture.
A 10 fold serial dilution using normal saline as diluents with 1g at soil & 0.1ml of 10-6 and 10-
7 dilution were spread on the plates in triplicates. The coliform forming links (CFU) of the
bacteria were counted after incubation at 28oC for 18-48 hour interval.
ENUMERATION AND ISOLATION OF HYDROCARBON UTILIZING BACTERIA
(HUB)
A mineral salt medium (Bushneg hass agar) using the vapour phase transfer method
determination of the hydrocarbon utilizing bacteria was used. The mineral salt medium was
solidified using 1.5% agar. Soil suspensions were prepared by 10 fold serial dilutions with 1g
of soil and 0.1m of 10-4 and 10-6 dilution was spread on the plates in triplicates. After
inoculation o the agar plates with the sample, a sterile filter paper (Whatman No.1)
saturated with crude oil was aseptically placed onto the inside of the lid (cover) of the Petri
dishes. The filter paper saturated with crude oil served as a sole carbon and energy source
for growth of the organisms on the surface through the vapour phase transfer. The plates
were then incubated in an inverted position at room temperature for 7 days after which the

average counts from triplicates were counted and recorded. (Abu and Chikere, 2006; R.B
Agbor et al, 2012)
BIOCHEMICAL TESTS, CHARACTERIZATION AND IDENTIFICATION OF
HYDROCARBON UTILIZING BACTERIA
Organisms with distinct colony characteristics that shows ability to utilize hydrocarbon in
the mineral salt medium were subsequently used for characterization and identifications for
the various set ups.
For characterization and identification of isolates in the various setup, the microscopy,
macroscopic, morphology, staining reaction, motility test, growth at 4oc and 41oc and
biochemical test were carried out according to Bergey’s Manual of Determinative
Bacteriology. (Alfred O Et al, 2011).

Dioxygenase activity was screened for by the inclusion of indole in the mineral salt medium
agar plates. Dioxygenases converts indole to indigo and the presence of blue colonies was
the selection criterion (Philip et al. 2005).
Colonies were sampled by filter lift from plates sprayed with catechol. The appearance of a
yellow pigment within 10 minutes of incubation at room temperature implies catechol 2, 3
dioxygenase activities. Catechol is an intermediate in hydrocarbon catabolism.
Changes in phospholipid profiles during the identification process were also examined.
STATISTICAL ANALYSIS
Data was subjected to statistical test to determine precision and accuracy and a logarithm
graph was plotted to determine the diversity and abundance of the oleophilic microbe
during the experiment
KEY DELIVERABLES
At the end of the experiment, the abundance and diversity of the oleophilic microbes will be
determined.

CONCLUSION
The increases in relative abundance and diversity of oleophilic microbes have a mark effect
in the biodegradation of total petroleum hydrocarbon. The ability of organisms working
together in bioremediation is highly specific and effective. Fertilizers can enhance these oil
loving microbes to stimulate a biodegradation process.
Regardless of the type of bioremediation used, bioremediation technology is a must
accepted.

REFERENCES
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Alfreda O. Nwadinigwe., Ekene G. Onyeidu. (2012). Bioremediation of crude oil- polluted soil
using bacteria and poultry manure monitored through soybean productivity.
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Frank Anayor Orji., Abiye Anthony Ibiene., Ekaette Nduka Dike. (2012). Laboratory scale
bioremediation of petroleum hydrocarbon-polluted mangrove swamp in the Niger
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Langley A., Gilbey M., Kennedy b. (2015). Analytical methods for the determination of total
petroleum hydrocarbon in soil. Environmental Protection and Heritage Council
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M. Vidali. Bioremediation. An overview. (2001). Pure applied chemistry. Pp 1169.
R.B, Agbor., I. A. Ekpo., A.N, Osuagwu.,U.U Udofia., E.C Okpako., S.P. Antai.(2012)
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