research proposal

EXECUTIVE SUMMARYINTRODUCTION

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.

Aggreh Erhovwon Peter

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

or flocculation

Non- directed

physic-

Cost effective Removal of heavy Metals


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

acceptor

All respiring bacteria require a terminal electron acceptor. In some cases,

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 PROBLEMThe effect of direct application of inorganic fertilizers to the environment when used as a bio

stimulant during a bioremediation process.

AIMThe 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 OBJECTIVESTo 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 SAMPLESThe 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 550 oc 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 30 oc 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.


http://www.agriculturesolutions.com/

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 MANURECOW 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 ANNALYSISCOW DUNG:

GOAT DROPPINGS:

COCONUT COIR DUST:

CASSAVA MESOCAP:

WHEAT:

PURIFICATION AND IDENTIFICATION OF HYDROCABON UTILIZING BACTERIADistinct 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 DELIVERABLESAt the end of the experiment, the abundance and diversity of the oleophilic microbes will be determined.


CONCLUSIONThe 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.


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