See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/326353074

Effect of Pig Manure on the Microbial Remediation of Crude Oil-Polluted Soil

Article in American Journal of Life Science Researches · April 2018

CITATIONS READS

4 467

3 authors, including:

Samuel Chinedu Onuorah Frederick John Chidi Odibo

Nnamdi Azikiwe University, Awka Nnamdi Azikiwe University, Awka
83 PUBLICATIONS 349 CITATIONS 85 PUBLICATIONS 930 CITATIONS

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

effect of palm oil mill effluent on the environment View project

Bioremediation of nitrates contaminating ground water View project

All content following this page was uploaded by Samuel Chinedu Onuorah on 12 July 2018.

The user has requested enhancement of the downloaded file.

American Journal of Life Science Researches
2018; 6(2): 76-90
Published online April, 2018 (http://www.diili.org/ojs-2.4.6/index.php/ajlsr/index)
ISSN: 2375-7485 (Print); ISSN: 2332-0206 (Online)

Original Paper

Effect of Pig Manure on the Microbial Remediation of Crude Oil-Polluted Soil

Onuorah Samuel1*, Soludo Christian1, Odibo Frederick1

Department of Applied Microbiology and Brewing Nnamdi Azikiwe University P.M.B. 5025 Awka, Nigeria
*Corresponding author: Onuorah Samuel Email Address: [email protected] Phone Number:
08061599174

ARTICLE INFO The effect of pig manure on the microbial remediation of crude oil-
Article history: polluted soil was studied using standard methods. The heterotrophic
Received 22 Feb. 2018 microorganisms isolated from the pristine soil, crude oil-polluted soil
Revised 22 Mar. 2018 and pig manure were Escherichia coli, Micrococcus luteus,
Accepted 12 Apr. 2018 Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus
aureus, Bacillus subtilis, Proteus vulgaris, Shigella flexneri, Serratia
marcescens, Flavobacterium rigense, Klebsiella aerogenes,
Arthrobacter oxydans, Aspergillus niger, Fusarium oxysporum,
Penicillium expansum, Cladosporium resinae, Candida utilis and
Trichoderma herbarum. All except Escherichia coli and
Staphylococcus aureus utilized crude oil as sole source of carbon and
energy for growth. The pH of the pristine soil was 7.6 but dropped to
7.1 after crude oil impaction and increased further to alkaline level
upon supplementation with pig manure after which it gradually
decreased during the course of the study. Addition of pig manure in
varying concentrations (10%,20%,30% and 40%) to the crude oil –
polluted soil caused an increase in the population of the heterotrophic
and hydrocarbon – utilizing micro-organisms and values of the
mineral nutrients. The physicochemical and microbiological
characteristics of the crude oil-amended soil were determined at the
end of fourteen weeks. The 40% amendment gave the highest
degradation of 84.6% while the unamended soil gave the lowest
degradation of 25.6%. As the amendment increased, the residual oil
content decreased. The ecotoxicity test showed that the bean seedlings
grew well in the amended crude oil-polluted soil but did not grow in
the unamended crude oil-polluted soil.
Keywords: pig manure, microbial
remediation, crude oil-polluted soil

Introduction
Petroleum hydrocarbons are subjected to a variety of natural processes such as
volatilization, biodegradation, photodecomposition, chemical oxidation, dispersion,
bioaccumulation, diffusion and leaching to ground water [1]. Petroleum hydrocarbon-
contaminated soils have been treated with the use of booms, skimmers, sorbents, in-situ
burning, dispersants and manual methods but these methods do not lead to a complete
clean-up of oil-polluted environments and may often lead to increased pollution of the
environment.
Onuorah et al., 2018

Bioremediation is the biotechnology which employs the catabolic activities of indigenous

hydrocarbon-utilizing micro organisms to decontaminate oil-polluted environments [2]. It
is environmentally friendly and cost effective and the techniques include natural
attenuation, biostimulation and bioaugumentation.
Cold regions have been considered to be especially sensitive to oil pollution because of the
prolonged degradation time of oil hydrocarbons [3]. Nevertheless, oil hydrocarbons are
also degraded by bacteria in cold environment [4], thus several psychotolerant bacteria
capable of degradation of oil hydrocarbons are known. Such microbial degradation enables
bioremediation either with or without intervention.
Some isolated organisms can effectively degrade single pollutant in laboratory conditions
but when introduced into actual field conditions with multiple pollutants, they cease to
function as anticipated [5]. In addition, the introduced strains may not compete well with
the indigenous micro organisms in the soil and remain dormant or unviable [6].
Bioremediation efficiency is therefore a function of the ability of the inoculated microbial
degraders to remain active in the natural environment [1].
Although petroleum is a source of abundant carbon, its lack of nitrogen and phosphorus
makes it less biodegradable than other biowastes such as waste food, sewage, sludge and
livestock manure [5]. Animal manure such as pig manure has been used as a fertilizer for
farming to improve soil aggregation, fertility, encourage soil microbial activity, promote
the soil trace mineral supply and improve plant nutrition, therefore in this work, the effect
of pig manure on the microbial remediation of crude oil-polluted soil was investigated.
MATERIALS AND METHODS
Samples Collection
The pristine soil samples were collected from a location at Ifite Awka, Nigeria with a
sterile hand trowel at a depth of five centimeters. They were pulled together and mixed
uniformly to give a composite sample which was thereafter introduced into a sterile glass
container and conveyed to the laboratory of Nnamdi Azikiwe University Awka for
analysis.
The pig manure was collected from Ausco farm in Awka, air dried, ground and stored in
the laboratory at room temperature for use while the crude oil was obtained from Eleme oil
field situated at Eleme Local Government Area of Rivers Sate, Nigeria and sterilized by
autoclaving at 121C for fifteen minutes before use.
Amendment of Pristine Soil
One kilogram of the composite pristine soil was polluted with one hundred milliliters of
sterile crude oil in a sterile glass vessel and distributed into five sterile conical flasks and
amended with varying concentrations of pig manure.
Conical flask A contained 70g of crude oil-polluted soil mixed with 8g of pig manure (10%
amendment).
Conical flask B contained 70g of crude oil-polluted soil mixed with 17g of pig manure
(20% amendment).
Conical flask C contained 70g of crude oil-polluted soil mixed with 30g of pig manure
(30% amendment).
Conical flask D contained 70g of crude oil-polluted soil mixed with 46g of pig manure
(40% amendment).
Conical flask E contained 70g of crude oil-polluted without pig manure (control). The
flasks were allowed to stand for fourteen weeks.

77
Effect of Pig Manure on the Microbial Remediation of Crude Oil-Polluted Soil

Determination of the physicochemical characteristics of the pristine soil, pig manure,

crude oil-polluted soil and amended crude oil-polluted soil.
Measurement of pH
The pH was measured using a 1:2 sample water ratio. Five grams of each of the samples
were dissolved with 10ml of distilled water in a beaker. The pH was measured with pH
water (JENWAY) that had standardized with pH buffers. The electrode was inserted into
the mixture and the value was recorded when the meter reading became stable.
Measurement of Nitrogen Content
This was carried out using the method of Subbaiah and Asija [7]. Twenty grams of each
sample were introduced into a 1 liter round bottom flask. Distilled water, 2 glass beads,
1ml of liquid paraffin, 100ml of potassium permanganate and 100ml of sodium hydroxide
solution were added to the flask. The mixture was distilled and the distillate was collected
in a beaker containing 20ml of boric acid working solution. The distillate was titrated with
standard 0.02N Sulphuric acid (H2SO4) till the colour changed from green to red and the
burette reading was recorded. The blank test was run without the samples.
Nitrogen (%) = A-B x Normality of H2SO4x0.014 x 100
Weight of Sample
Where A = Volume of standard H2SO4 required for sample
B = Volume of standard H2SO4 required for blank.

Measurement of Phosphorus Content

The method used by Olsen and Sommers [8] was adopted. 2.5g of the sample were
weighed into a 150ml conical flask. 0.3g of phosphate-free activated charcoal and 50ml of
Olsen reagent were added and the mixture was thereafter shaken for 20 minutes on a shaker
at 180rpm. The mixture was filtered through a filter paper and 5ml aliquot were transferred
into a 25ml volumetric flask. 4ml of freshly prepared ascorbic acid and ammonium
molybdate solution were added. The mixture was shaken and kept for 30 minutes. A
standard curve was prepared using 0,1,2,3,4 and 5mls of standard phosphorus solution. The
absorbance and colour intensity were measured after 30 minutes at 882mm. The blank
sample was run with the extracting solution.
Phosphorus (%) = GRx50x5 x 100
Weight of sample
Where GR = Concentration of Phosphorus in the analysed sample read from the standard
curve.
Measurement of Calcium and Magnesium Together
The calcium and magnesium contents (combined) of the samples were measured as done
by El-Mahi et al [9]. Two grams of the sample were introduced into a conical flask, 30ml
of ammonium acetate were added to it and the flask was shaken for five minutes and
decanted. 30ml of 0.5N hydrochloric acid were added and the mixture was agitated for five
minutes in an upright position. The solution was filtered by passage through a Whatman
No.1 fitter paper and the filtrate was collected. 20ml of the filtrate were pipetted out into a
150ml conical flask and 50ml of distilled water were added to it. 10ml of buffer solution,
10 drops each of NH2OH.HCL, Potassium ferrocyanide, Triethanolamine (TEA) and
Eriochrome black T indicator were added and the mixture was titrated with a standard
Ethylene diamine tetra acetic acid (EDTA) to a permanent blue colour.

78
Onuorah et al., 2018

Measurement of Calcium Content Alone

The method used by El-Mahi et al. [9] was adopted. Five millilitres of the filtrate were
pipetted out. Ten drops each of NH2OH.HCL, Potassium ferrocyanide and Triethanolamine
and three drops of 10% Sodium hydroxide solution were added to raise the pH to 12. Five
drops of Calcon indicator were added and the mixture was titrated against EDTA till there
was a colour change from red to blue.
Measurement of Magnesium Content Alone
This was measured using the method of El-Mahi et al. [9]. The magnesium content
was determined from the calculation:
Ca + Mg together – Ca alone = Mg
Measurement of Organic Carbon Content
This was performed using the method of Walkley [10]. One gram of the ground
sample was introduced into a dried 500ml volumetric flask. Ten milliliters of 0.167%
potassium dichromate solution were added and the mixture swirled gently till the sample
and the reagents were mixed and thereafter vigorously. 20ml of concentrated sulphuric acid
were added rapidly and the flask swirled for one minute. The reaction was allowed to
proceed for 30 minutes on asbestos sheet. 200ml of distilled water, 10ml of concentrated
orthophosphoric acid, 0.2g of sodium fluoride and 1ml of ferroin indicator solution were
added to the flask and the excess dichromate was titrated with 0.5N ammonium ferrous
sulphate till a brownish-red colour was observed. A blank test was run simultaneously
without the sample.
Organic Carbon (%) = (B-S) X N X 0.0003 X 100
Weight of Sample
Where B= Volume of standard 0.5N ammonium ferrous sulphate required for blank
S= Volume of standard 0.5N ammonium ferrous sulphate required for sample
N= Normality of standard 0.5N ammonium ferrous sulphate.
Determination of the microbiological characteristics of the pristine soil, pig manure,
crude oil-polluted soil and amended crude oil-polluted soil.

Isolation of total heterotrophic bacteria

One gram of the sample was serially-diluted using distilled water and one milliliter of the
serially-diluted sample (105) was plated out on sterile nutrient agar plate containing
0.05mg/ml of Ketoconazole to inhibit fungal growth. Duplicate plates were prepared and
incubation was carried out in an inverted position at 30oC for 48 hours. Colonies that
developed after incubation were counted and the total heterotrophic bacteria expressed as
colony forming units per gram of sample.
Isolation of total fungi
One gram of the sample was diluted serially using distilled water and one milliliter aliquot
of the serially-diluted sample (105) was spread-plated on Sabouraud dextrose agar
containing 0.05mg/ml of chloramphenicol to inhibit bacterial growth. Duplicate plates were
prepared which were incubated at 300C for 72 hours in an inverted position. The colonies
that grew after incubation were counted and the total fungi expressed as colony forming
units per gram of the sample.
Isolation of hydrocarbon-utilizing bacteria
One gram of the sample was serially-diluted with distilled water and one milliliter aliquot
of the serially-diluted sample was plated out on nutrient agar containing nystatin at a
concentration of 0.05mg/ml to inhibit fungal growth. Sterile filter paper impregnated with
crude oil was placed on the cover of the Petri dish to supply hydrocarbons to the organisms
79
Effect of Pig Manure on the Microbial Remediation of Crude Oil-Polluted Soil

through the vapor transfer phase. Duplicate plates was prepared and incubated at 30oC for
48 hours after which the colonies that developed were counted and the hydrocarbon-
utilizing bacteria expressed as colony forming units per gram of the sample.
Isolation of hydrocarbon-utilizing fungi
One gram of each sample was serially-diluted using distilled water and one milliliter of it
was spread-plated on Sabouraud dextrose agar which contained 0.05mg/ml of
chloramphenicol to inhibit bacterial growth. Sterile filter paper saturated with crude oil was
placed on the cover of the Petri dish to supply hydrocarbons to the organisms in the sample
through the vapour transfer phase. Duplicate plates were prepared per sample and
incubated at 30oC for 72 hours. Colonies that developed after incubation were counted and
the hydrocarbon-utilizing fungi expressed as colony forming units per gram of the sample.
Characterization and Identification of the Microbial Isolates
The bacterial isolates were characterized morphologically, biochemically and molecularly.
Molecular identification was based on 16SrDNA sequencing using FASTA algorithm. The
tests carried out were Gram staining, motility, catalase, coagulase, citrate utilization,
indole, oxi dase, methyl red, spore, voges proskaeur and sugar fermentation tests. They
were identified using Bergeys manual of Determinative Bacteriology.
The fungi was characterized using the lactophenol cotton blue staining, slide culture,
urease, motility, germ tube and sugar fermentation tests as done by Onuorah et al [11].
They were identified using the fungal atlas.
Gram staining
The test bacterium was smeared on a clean grease-free slide and fixed by passage over
Bunsen flame. The slide was thereafter flooded with crystal violet solution, allowed to
penetrate for 60 seconds and washed off with tap water. It was next flooded with lugol’s
iodine solution and washed off with tap water after being allowed to stay for 60 seconds.
The slide was thereafter decolourized with acetone and left for 10 seconds before being
rinsed with tap water. The smear was then flooded with safranin solution, allowed to stand
for 60seconds, rinsed with tap water and allowed to dry in air after which the slide was
examined under the oil immersion lens of the microscope. Gram positive bacteria stained
purple while the negative ones were red in colour.
Motility test
A semi-solid nutrient agar was used. It was stab-inoculated with the test organism with a
sterile straight wire, incubated at 37oC for 24 hours and observed for motility. Motile
organisms grew away from the line of inoculation while the non-motile ones grew along
the line of the stab-inoculation.
Catalase test
A loopful of the test bacterium was introduced into a test tube containing 3% hydrogen
peroxide. The production of gas bubbles was a positive reaction while the absence of gas
bubbles was a negative result.
Coagulase test
A drop of distilled water was placed on a clean slide and the test bacterium was emulsified
in it. One milliliter of human plasma was added and gently mixed. The slide was observed
for clumping of the bacterium within 10 seconds which indicated a positive reaction.
Citrate utilization test
The test bacterium was streaked on the surface of Simmon citrate agar and incubated at
37oC for 24hours. The change in colour of the medium from green to blue was a positive
result.

80
Onuorah et al., 2018

Indole test
The test bacterium was inoculated into a test tube containing peptone water which was
thereafter incubated at 37oC for 24 hours. Two drops of Kovac’s reagent were added to the
medium which was thereafter observed for the production of a pink-red colour which
indicated a positive reaction.
Oxidase test
Five drops of oxidase reagent were introduced on a filter paper. A smear of the test
bacterium was made on the filter paper. The production of a deep purple colour within
10seconds was a positive result.
Methyl red test
A sterile tube containing glucose phosphate peptone water was inoculated with the test
bacterium and incubated at 37oC for 48 hours. Five drops of methyl red solution were
thereafter added to the medium which was gently shaken and allowed to stand. The
production of a red colour was a positive reaction.
Spore test
A film of the test bacterium was prepared on a clean slide, air-dried and fixed with minimal
flaming over a Bunsen flame. The slide was thereafter placed on the rim of a beaker of
boiling water with the film uppermost and flooded with 0.5% aqueous solution of
malachite green when large droplets had condensed on it’s underside and left to stand for
five minutes while the water continued to boil. The slide was then rinsed with clean water,
treated with 0.5% safranin solution and left to stand for 30 seconds before being rinsed
with water. The slide was after that air-dried and viewed under the microscope.
Voges Proskaeur test
The test bacterium was inoculated into a sterile test tube containing glucose phosphate
peptone water and incubated at 37oC for 48 hours after which five drops of Baritt A (alpha-
naphtol) and Baritt B (Potassium hydroxide) reagents were added. The production of a
pink-burgundy colour was a positive result.
Sugar fermentation test
Two drops of bromothymol blue solution and one milliliter of a 1% solution of the test
sugar was introduced into a flask containing sterile peptone water. The mixture was
distributed in 5ml amounts into test tubes containing Durham’s tubes and autoclaved. Upon
cooling, the tubes was inoculated with the test bacterium singly and incubated at 37 oC for
48 hours. A change in the colour of the medium to yellow denoted acid production and a
positive result while the formation of a void in the Durham’s tubes showed gas production
and a positive reaction. The sugars used were glucose, sucrose, fructose and lactose.
Lactophenol cotton blue staining
The test mould was gently mixed with a drop of lactophenol cotton blue solution on a clean
glass slide. The slide was after that covered with a cover slip and viewed under the
microscope.
Slide culture test
The test mould was inoculated on sterile Sabouraud dextrose agar plate and a cover slip
was placed beside the mould. The medium was thereafter incubated in an inverted position
at 37oC for five days after which the cover slip was removed and placed on a slide
containing a drop of lactophenol cotton blue solution. The slide was after that viewed under
the microscope.

81
Effect of Pig Manure on the Microbial Remediation of Crude Oil-Polluted Soil

Urease test
Sterile Urea agar in McCartney bottle was inoculated with the test yeast and incubated for
seven days at 37oC. The production of light pink to light yellow colour was a positive
result.
Germ tube test
A test tube containing 0.5ml of human serum was inoculated with the test yeast and
incubated at 37oC for three hours after which a drop of the serum-yeast culture was placed
on a slide, covered with cover slip and examined under the microscope for the presence of
tube-like outgrowths from the cell.
Screening test for hydrocarbon utilization by the microbial isolates
The mineral salts medium described by Olukunle [12] was used. It was introduced into test
tubes in 9ml amounts. One milliliter of crude oil was added to each of the test tubes which
was thereafter capped, sterilized by autoclaving and allowed to cool. Upon cooling, the test
tubes were inoculated with the microbial isolates individually. An uninoculated tube was
used as the control. The tubes were incubated at 30oC for fourteen days in a rotary shaker at
180rpm after which they were observed for turbidity for the bacterial isolates and clarity
for the fungal isolates.
Residual hydrocarbon test
The content of each flask was sterilized by autoclaving at 121oC for fifteen minutes at the
end of the fourteen weeks to destroy all microorganisms and the residual crude oil content
was determined by the method of Amadi [13].
The content of each flask was transferred into a separating funnel and the residual crude oil
extracted with toluene. The funnel was cocked tightly and tilted for five minutes. The
flasks were shaken, occasionally refluxed to let out air and left to stand for 30 minutes.
The toluene crude oil mixture was collected in a sample bottle and analyzed
spectrophotometrically by diluting the mixture with a known volume of toluene and
reading the absorbance at 420nm. A standard graph was prepared using known volume of
crude oil.
The residual crude oil content was determined from the formular GR X IVS
WT

Where WT= Weight of soil sample

IVS= Initial volume of toluene used for the extraction
GR = absorbance reading at 420nm x slope of the standard graph
% Residual Crude oil content = Crude oil recovered X 100
Crude oil applied

% Reduction of Crude oil = Crude oil recovered – Crude oil applied X 100
Crude oil applied

Ecotoxicity test
Soil samples were polluted with crude oil (100ml) and amended with varying
concentrations of pig manure (10%, 20%, 30% and 40%). Bean seeds were cultivated on
each of the amended soil as well as the unamended soil. Growth parameter (shoot height
and root length) was measured at the end of fourteen weeks.
The plants were carefully uprooted and washed in tap water to flush out the soil particles.
The shoot height and the root length were measured in centimeter using a meter rule.

82
Onuorah et al., 2018

Statistical analysis
Pearson’s statistical analysis was carried out to determine the correlation between the
various treatment options and the residual crude oil content of the polluted soil.

RESULTS
The physicochemical and microbiological characteristics of the pristine soil, crude oil-
polluted soil and pig manure are shown in Table 1. The samples were alkaline and
contained nitrogen, phosphorus, calcium, magnesium and organic carbon which are
essential for the growth of plants, animals and microorganisms. In addition, they contained
bacteria and fungi including the hydrocarbon-utilizing species.
Table 1: Physicochemical and microbiological characteristics of the pristine soil, crude oil-polluted soil and
pig manure
Characteristics Pristine soil Crude oil-polluted soil Pig manure
pH 7.6 7.1 8.0
Nitrogen (%) 1.4 1.6 2.4
Phosphorus (%) 0.7 0.8 1.3
Calcium (%) 1.4 1.6 2.3
Magnesium (%) 0.3 0.4 0.8
Organic carbon (%) 0.7 0.9 2.8
Total heterotrophic bacteria (cfu/g) 5.3x105 5.5x105 6.5x105
Total fungi (cfu/g) 3.8x105 4.0x105 4.4x105
Hydrocarbon utilizing bacteria (cfu/g) 3.0x105 3.3x105 3.7x105
Hydrocarbon utilizing fungi (cfu/g) 2.2x105 2.5x105 3.0x105
Several heterotrophic microorganisms were isolated from the samples as presented in Table
2. They were Escherichia coli, Micrococcus luteus, Enterococcus faecalis, Staphylococcus
aureus, Proteus vulgaris, Shigella flexneri, Serratia marcescens, Pseudomonas aeruginosa,
Bacillus subtilis, Flavobacterium rigense, Klebsiella aerogenes, Arthrobacter oxydans,
Aspergillus niger, Fusarium oxysporum, Penicillium expansum, Cladosporium resinae,
Candida utilis, Candida tropicilis and Trichoderma herbarum.
Table 2: Heterotrophic microorganisms isolated from the pristine soil, crude oil-polluted soil and pig manure
Microorganisms Pristine soil Crude oil-polluted soil Pig manure
Escherichia coli + - +
Micrococcus luteus + + +
Enterococcus faecalis + - +
Staphylococcus aureus + - +
Proteus vulgaris - + +
Shigella flexneri - - +
Serratia marcescens + + +
Pseudomonas aeruginosa + + +
Bacillus subtilis + + -
Flavobacterium rigense - + -
Klebsiella aerogenes - + -
Arthrobacter oxydans - + -
Aspergillus niger + + +
Fusarium oxysporum + + -
Penicillium expansum + + +
Cladosporium resinae + + +
Candida utilis - + -
Candida tropicalis - - +
Trichoderma herbarum - - +
+ detected - not detected
All the organisms except Escherichia coli and Staphylococcus aureus were identified as
crude oil-utilizing microorganisms as shown in Table 3.

83
Effect of Pig Manure on the Microbial Remediation of Crude Oil-Polluted Soil

Table 3: Crude oil-utilizing microorganisms isolated from the pristine soil, crude oil-polluted soil and pig
manure
Microorganisms Pristine soil Crude oil-polluted soil Pig manure
Micrococcus luteus + + +
Enterococcus faecalis + - +
Proteus vulgaris - + +
Shigella flexneri - - +
Serratia marcescens + + +
Pseudomonas aeruginosa + + +
Bacillus subtilis + + -
Flavobacterium rigense - + -
Klebsiella aerogenes - + -
Arthrobacter oxydans - + -
Aspergillus niger + + +
Fusarium oxysporum + + -
Penicillium expansum + + +
Cladosporium resinae + + +
Candida utilis - + -
Candida tropicalis - - +
Trichoderma herbarum - - +

+ detected - not detected

Table 4 showed the crude oil-utilization potential of the hydrocarbon-utilizing

microorganisms from the samples. Pseudomonas aeruginosa and Bacillus subtilis had the
highest utilization potential (+++) among the bacterial isolates while Aspergillus niger and
Penicillium expansum had the highest utilization potential (+++) among the fungal isolates.

Table 4: Crude oil-utilization potential of hydrocarbon-utilizing microbial isolates from the pristine soil,
crude oil-polluted soil and pig manure
Hydrocarbon utilizing microbial isolates Crude oil utilization
Micrococcus luteus ++
Enterococcus faecalis ++
Proteus vulgaris ++
Shigella flexneri +
Serratia marcescens ++
Pseudomonas aeruginosa +++
Bacillus subtilis +++
Flavobacterium rigense ++
Klebsiella aerogenes +
Arthrobacter oxydans ++
Aspergillus niger +++
Fusarium oxysporum +
Penicillium expansum +++
Cladosporium resinae ++
Candida utilis ++
Candida tropicalis ++
Trichoderma herbarum ++
+ minimal utilization ++moderate utilization +++heavy utilization

84
Onuorah et al., 2018

The physicochemical and microbiological characteristics of the crude oil-polluted soil upon
amendment with pig manure are shown in Table 5. There was an increase in the values of
the physicochemical and microbiological characteristics.

Table 5: Physicochemical and microbiological characteristics of the crude oil-polluted soil upon amendment
with pig manure
Characteristics Crude oil- Crude oil- Crude oil- Crude oil-
polluted soil polluted soil polluted soil polluted soil
+10% pig +20% pig +30% pig +40% pig
manure manure manure manure
pH 7.5 7.6 7.8 8.3
Nitrogen (%) 1.8 2.0 2.4 3.2
Phosphorus (%) 0.9 1.0 1.2 1.4
Calcium (%) 2.0 2.2 2.6 3.0
Magnesium (%) 0.6 0.7 0.9 1.0
Organic carbon (%) 1.1 1.6 2.0 3.6
Total heterotrophic 5.8x105 6.0x105 6.5x105 7.2x105
bacteria (cfu/g)
Total fungi (cfu/g) 4.2x105 4.4x105 4.9x105 5.3x105
5 5 5
Hydrocarbon utilizing 3.8x10 4.3x10 4.8x10 5.1x105
bacteria (cfu/g)
Hydrocarbon utilizing 2.7x105 3.0x105 3.5x105 3.8x105
fungi (cfu/g)

Table 6 showed the physicochemical and microbiological characteristics of the crude oil-
polluted soil at fourteen weeks of amendment with pig manure. There was a decrease in the
physicochemical characteristics of the amended soil sample including the control soil while
the microbiological characteristics increased in both the amended and control soil samples.
However, the decrease in the physicochemical characteristics as well as the increase in the
microbiological characteristics of the control sample was lower than that of the amended
soil samples.
Table 6: Physicochemical and microbiological characteristics of the crude oil-polluted soil at fourteen weeks
of amendment with pig manure
Characteristics Crude oil- Crude oil- Crude oil- Crude oil- Control
polluted soil polluted soil polluted soil polluted soil
+10% pig +20% pig +30% pig +40% pig
manure manure manure manure
pH 6.6 6.6 6.4 6.2 6.8
Nitrogen (%) 0.9 1.0 1.1 1.7 1.2
Phosphorus (%) 0.6 0.5 0.6 0.7 0.6
Calcium (%) 1.1 1.2 1.3 1.5 1.2
Magnesium (%) 0.4 0.4 0.5 0.5 0.2
Organic carbon (%) 0.2 0.4 0.6 1.1 0.5
Total heterotrophic 6.3x105 6.7x105 7.5x105 9.0x105 5.7x105
bacteria (cfu/g)
Total fungi (cfu/g) 4.8x105 5.2x105 5.7x105 6.6x105 4.0x105
5 5 5 5
Hydrocarbon 4.2x10 4.8x10 5.5x10 6.1x10 3.5x105
utilizing bacteria
(cfu/g)
Hydrocarbon 3.0x105 3.4x105 3.9x105 4.2x105 2.4x105
utilizing fungi
(cfu/g)

85
Effect of Pig Manure on the Microbial Remediation of Crude Oil-Polluted Soil

The residual hydrocarbon content of the amended and unamended crude oil-polluted soil is
presented in Table 7. The percentage degradation was highest in the soil amended with
40% pig manure (84.6%) while the unamended soil had the lowest percentage degradation
(25.6%)

Table 7: Residual hydrocarbon content of the amended and unamended crude oil-polluted soil
Amendment with pig Initial hydrocarbon Residual hydrocarbon %
manure content (ml) content (ml) Degradation
10% 1.00 0.426 57.4
20% 1.00 0.388 61.2
30% 1.00 0.267 73.3
40% 1.00 0.154 84.6
Unamended soil 1.00 0.744 25.6

The in-situ and ex-situ shoot length of the bean seedlings grown in the amended and
unamended crude oil-polluted soil are shown in Table 8. The seedlings grown both in-situ
and ex-situ in the soil amended with 40% pig manure had the longest shoot length while
the seeds grown in the unamended soil did not germinate.
Table 8: In-situ and Ex-situ shoot length of the bean seedlings grown in the amended and unamended crude
oil-polluted soil
Amendment with pig manure In situ shoot length (cm) Ex situ shoot length (cm)
10% 5.10 7.20
20% 9.70 11.60
30% 13.40 15.70
40% 19.30 21.50
Unamended soil 0.00 0.00

Table 9 showed the in-situ and ex-situ root lengths of the bean seedlings grown in the
amended and unamended crude oil-polluted soil. The seeds did not germinate in the
unamended soil while the seedlings grown in the crude oil-polluted soil amended with 40%
pig manure had the longest root length both in-situ and ex-situ.
Table 9: In-situ and Ex-situ root length of the bean seedlings grown in the amended and unamended crude
oil-polluted soil
Amendment with pig manure In situ root length (cm) Ex situ root length (cm)
10% 3.20 4.30
20% 5.70 7.60
30% 7.50 9.40
40% 8.90 11.70
Unamended soil 0.00 0.00

DISCUSSION
The physicochemical and microbiological analysis of the pristine soil, crude oil-polluted
soil and pig manure showed that the samples contained nitrogen, phosphorus, calcium,
magnesium, organic carbon and several heterotrophic and hydrocarbon-utilizing
microorganisms in varying proportions (Table1). The pH of the samples was alkaline. The

86
Onuorah et al., 2018

introduction of crude oil caused a slight increase in the mineral nutrients with a significant
increase in the heterotrophic and hydrocarbon-utilizing microorganisms.
These observations agreed with the report of Onyeagba et al [14] who studied the effects of
pig and chicken waste compost on physicochemical and biological indices of petroleum
contaminated Orashi-River wetland in Egbema, Southern Nigeria; Agarry et al [6] who
worked on the bioremediation of soil artificially contaminated with petroleum hydrocarbon
oil mixtures: evaluation of the use of animal manure and chemical fertilizer and Udebuani
et al [15] who studied the value of animal manure in the enhancement of bioremediation
processes in petroleum hydrocarbon contaminated agricultural soil.
Several heterotrophic microorganisms were isolated from the pristine soil, crude oil-
polluted soil and the pig manure (Table 2). They were Escherichia coli, Micrococcus
luteus, Enterococcus faecalis, Staphylococcus aureus, Proteus vulgaris, Shigella flexneri,
Serratia marcescens, Pseudomonas aeruginosa, Bacillus subtilis, Flavobacterium rigense,
Klebsiella aerogenes, Arthrobacter oxydans, Aspergillus niger, Fusarium oxysporum,
Penicillium expansum, Cladosporium resinae, Candida utilis, Candida tropicalis and
Trichoderma herbarum.
This result conformed to the report of Udebuani et al [15]; Yakubu [16] who worked on the
biodegradation of Lagoma crude oil using Pig dung; Bento et al [17] in their study of the
comparative bioremediation of soils contaminated with diesel oil by natural attenuation,
biostimulation and bioaugumentation; franco et al [18] in their study on the microbiological
resilience of soils contaminated with crude oil; Omotayo et al [19] who studied the
degradation of aviation fuel by microorganisms isolated from tropical polluted soils;
Stephen and Panneerselvam [20] who investigated the prevailing fungi on the hydrocarbon-
polluted soil and Chikere and Azubike [21] in their work on the characterization of
hydrocarbon utilizing fungi from hydrocarbon polluted sediments and water.
All the microbial isolates from the pristine soil, crude oil-polluted soil and pig manure
except Escherichia coli and Staphylococcus aureus were found to be crude oil utilizers
(Table 3). This result agreed with the reports of Bento et al [17]; Chikere et al [22] in their
assessment of the bioreactor-based bioremediation of hydrocarbon-polluted Niger Delta
marine sediment, Nigeria; Chikere and Ekwuabu [23] in their study of the culture-
dependent characterization of hydrocarbon utilizing bacteria in the selected crude oil-
impacted sites in Bodo, Ogoniland, Nigeria; Omotayo et al [19]; Yakubu [16]; Chikere and
Azubike [21] and Stephen and Panneerselvam [20].
The hydrocarbon utilizing microorganisms from the pristine soil, crude oil-polluted soil
and pig manure produced varying degree of turbidity for the bacteria and clarity for the
fungi while growing in the mineral salts oil medium (Table 4). Pseudomonas aeruginosa
and Bacillus subtilis produced the highest turbidity (+++) among the bacterial isolates
while Aspergillus niger and Penicillium expansum produed the highest clarity among the
fungal isolates. This result agreed with the finding of Yakubu [16] and Chikere and
Ekwuabu [23].
There was an increase in the values of the mineral nutrients, the heterotrophic and the
hydrocarbon utilizing microorganisms upon the amendment with varying concentration of
pig manure (Table 5). This increase may be attributed to the microorganisms in the pig
manure as well as the physicochemical constituents. This result conformed to the report of
Onyeagba et al [14].
The physicochemical and microbiological analysis of the crude oil polluted soil samples at
fourteen weeks of amendment with pig manure showed that there was a decrease in the
values of the mineral nutrients (Table 6). The microbial population also increased while the
87
Effect of Pig Manure on the Microbial Remediation of Crude Oil-Polluted Soil

pH values decreased to the slightly acidic level. This increase in the microbial population
may be attributable to the utilization of the mineral nutrients in both the crude oil-polluted
soil and the pig manure as carbon and energy sources by the microbial isolates, hence the
decrease in the values of such nutrients. These organisms probably produced some acidic
metabolites that led to the decrease in the pH values. This result also agreed with the
findings of Yakubu [16] and Okpokwasili and Okorie [24].
There was a decrease in the initial crude oil content at fourteen weeks of amendment with
pig manure (Table 7). The amendment with 40% pig manure had the highest degradation of
the crude oil of 84.65%. There was also a reduction in the initial crude oil content of the
unamended soil (25.6%) indicating that natural attenuation is a also useful technique for the
clean-up of oil-polluted environment though at a slower rate. The decrease in crude oil
content could be attributed to the enhanced utilization of hydrocarbons by microbial
isolates. This result agreed with the previous works carried out by Adieze et al [25].
An evaluation of the effectiveness of bioremediation of the amended crude oil-polluted soil
was carried out in situ and ex situ using bean seeds as monitoring tool. There was
germination in the soils amended with pig manure which indicated that the fertility of the
soil was restored with the pig manure amendment while the seeds did not germinate in the
unamended soil indicating that the crude oil rendered the unamended soil infertile. The ex-
situ shoot and root lengths were higher than the in-situ shoot and root lengths. This could
be attributed to the laboratory conditions the in-situ experiment was carried out.
The 40% pig manure amendment had the highest length of the shoot and roots both in-situ
and ex-situ (Tables 8 and 9) indicating that as the amendment with pig manure increased,
the crude oil-polluted soil became better remediated, hence the better growth of the bean
seeds. This result conformed to the report of Ekpo and Nya [26] on the effects of
amendments with poultry manure on diesel oil-polluted soil on germination and growth
performance of some forest tree species. The seedlings grew well in the amended crude oil-
polluted soil indicating that the pig manure had positive effects on the microbial
remediation of the crude oil-polluted soil.

CONCLUSION

An understanding of microbial degradation and bioremediation process could assist to

achieve the remediation of oil-polluted lithospheric environment. Bioremediation was
achieved in less time with biostimulation as compared to natural attenuation. This study
showed that crude oil-polluted soil can have adverse effect on germination and growth
performance of crops but such soil can be remedied by the addition of organic nutrients
especially pig manure to enhance it’s fertility. Pig manure is therefore recommended for
the bioremediation of crude oil-polluted soil.

REFERENCES
1. M. Alexander. Aging, bioavailability and overestimation of risk from environmental
pollutants. Environmental Science and Technology. Vol. 34, No. 20, 4259-4265, 2000.
DOI: 10.1021/es001069
2. R.M. Atlas, R. Bartha. Microbial Ecology: Fundamentals and Application (4th edition).
Benjamin Cummings, San Francisco, California, U.S.A. Pp 523-530, 1998.
3. A.J. Lin, X.H. Zhang, Y.H.SU, Y.HU, Q Cao, Y.G. Zhu. Chemical fixation of metals in
soil using bone char and assessment of the soil genotoxicity. Journal of Natural Science.
Vol. 28, No. 2, 232-237, 2007.
88
Onuorah et al., 2018

https:// www.ncbi.nlm.nih.gov>pubmed

4. R. B. Meagher, Phytoremediation of toxic elemental and organic pollutants. Current

Opinion in Plant biology. Vol. 3, No. 2, 153-162, 2000
https://doi.org/10.1016/S 1369-5266 (99) 00054-0
5. R.M. Atlas. Bioremediation of petroleum pollutants. International Biodeterioration and
Biodegradation. Vol. 35, No. (1-3), 317-327, 1995.
www.sciencedirect.com>article>pii
6. S.E Agarry, N.C. Owabor, R.O. Yusuf. Bioremediation of soil artificially contaminated
with petroleum hydrocarbon oil mixtures: evaluation of the use of animal manure and
chemical fertilizer. Bioremediation Journal. Vol. 14, No. 4, 189-195, 2010.
http://dx.doi/org/10.1080/10889868.2010.514965
7. B.V. Subbiah, G.L. Asija. A rapid procedure for the determination of available nitrogen
in soil. Current Science. Vol.25, No.8, 259-260, 1956.
www.Sciepub.com/reference/79523.
8. S.R Olsen, L.E. Sommers. Phosphorus. In. A.L. Page and R.H. Miller (Eds). Methods of
soil analysis, part 2, 2nd edition, Agronomy Monograph 9, American Society of
Agronomy and Soil Science Society of America, Madison, Pp 403-430, 1982.
9. Y.E EL-Mahi, I.S. Ibrahim, H.M. Abel-Majid, A.M.A. Eltilib. A simple method for the
estimation of calcium and magnesium carbonates in soils. Soil Society of America
Journal. Vol.51, No.5, 1152-1155, 1987.
DOI: 10.2136/sssaj1987.03615995005100050010x
10. A. Walkley. A critical examination of a rapid method for determining organic
carbon in soils-Effects of variations in digestion conditions and inorganic soil
constituents. Soil Science. Vol.63, 251-264, 1947.
DOI: 10.1097/00010694-194704000-00001
11. Onuorah Samuel, Soludo Christian, Odibo Frederick. Biodeterioration potentials of
microorganisms isolated from pig manure obtained at Awka, Nigeria. Natural Resources
and Conservation. Vol.5, No.3, 33-43, 2017.
DOI: 10.13189/nrc.2017.050301
12. O.F. Olukunle. Characterization of indigenous microorganisms associated with
crude oil-polluted soils and water using traditional techniques. Microbiology Journal.
Vol.3, 1-11, 2013.
DOI: 10.3923/mj.2013.1.11
13. A. Amadi. An assessment of the performance of some petroleum hydrocarbon
degrading microorganisms in aqueous axenic cultures. Discovery and Innovation. Vol.4,
No.4, 61-67, 1992
africabib.org
14. R.A. Onyeagba, V.C. Nwaugo, C. Asamudu, J.O. Nduka. Effects of pig and
chicken wastes compost on physicochemical and biological indices of petroleum
contaminated Orashi-River wetland in Egbema, Southern Nigeria. ABSU Journal of
Environment, Science and Technology. Vol.1, 1-13, 2011
https://www.google.com.ng/search?
15. A.C. Udebuani, C.I. Okoli, H.C. Nwigwe, P.T.E. Ozoh. The value of animal
manure in the enhancement of bioremediation processes in petroleum hydrocarbon
contaminated agricultural soils. Journal of Agricultural Technology. Vol.8, No.6, 1935-
1952, 2012.
http://www.ijat-aatsea.com
89
 Effect of Pig Manure on the Microbial Remediation of Crude Oil-Polluted Soil

16. M.J Yakubu. Biodegradation of Lagoma crude oil using Pig dung. African Journal
of Biotechnology. Vol.6, No.24, 2821-2825, 2007.
www.Sciepub.com/reference /162397
17. F.M. Bento, F.A.O Camargo, B.C. Okeke, W.T. Frankenberger. Comparative
bioremediation of soils contaminated with diesel oil by natural attenuation,
biostimulation and bioaugumentation. Bioresource Technology. Vol.96, No.9, 1049-
1055, 2005.
https://doi.org/10.1016/j.biortech.2004.09.008
18. I. Franco, M. Contin, G. Bragato, M. De-Nobili. Microbiological resilience of soils
contaminated with crude oil. Geoderma. Vol.121, No.(1-2), 17-30, 2004.
https://doi.org/10.1016/j.geoderma.2003.10.002
19. A.E Omotayo, A.O Efetie, G.Oyetibo, M.O. Ilori, O.O. Amund. Degradation of
aviation fuel by microorganisms isolated from tropical polluted soils. International
Journal of Biological and Chemical Sciences. Vol.5, No.2, 698-708, 2011.
DOI: 10.4314/ijbcs.v5i2.72133
20. M. Stephen, A. Panneerselvam. Study of the prevailing fungi on the hydrocarbon
polluted soil. World Journal of Pharmaceutical Research. Vol.4, No.2, 1102-1108, 2015
www.wjpr.net
21. C.B. Chikere, C.C. Azubike. Characterization of hydrocarbon-utilizing fungi from
hydrocarbon-polluted sediments and water. Nigerian Journal of Biotechnology. Vol.27,
49-54, 2014.
http://www.ajol.info/index.php/njb/index
22. C.B. Chikere, B.O. Chikere, G.C. Okpokwasili. Bioreactor-based bioremediation
of hydrocarbon-polluted Niger Delta marine sediment Nigerian Journal of
Biotechnology. Vol.2, No.1, 53-66, 2012.
www.Sciepub.com.reference
23. C.B. Chikere, C.B. Ekwuabu. Molecular characterization of autochthonous
hydrocarbon utilizing bacteria in oil-polluted sites at Bodo Community, Ogoniland,
Niger Delta, Nigeria. Nigerian Journal of Biotechnology. Vol.27, No.1, 23-45, 2014.
https://www.ajol.info>njb>article>view
24. G.C. Okpokwasili, B.B. Okorie. Biodeterioration potentials of microorganisms
isolated from car engine lubricating oil. Tribology international. Vol.21, No.4, 215-220,
1988
DOI: 10.1016/0301-679 X (88) 90020-5
25. I.E. Adieze, R.N Nwabueze, G.O.C. Onyeze. Effect of poultry manure on the
microbial utilization of hydrocarbon in oil-polluted soil. Nigerian Journal of
Microbiology. Vol.17, No.1, 12-16, 2003.
https://www.researchgate.net/publication/232632133
26. F.E. Ekpo, E.J. Nya. Effect of poultry manure amendments on diesel oil-polluted
soil on germination and growth performance of some forest tree species. Journal of
Research in Environmental Science and Toxicology. Vol.1, No.7, 195-200, 2012.
http://www.interesjournal

90

View publication stats