Content uploaded by Renner Nrior
Author content
All content in this area was uploaded by Renner Nrior on Mar 09, 2021
Content may be subject to copyright.
_____________________________________________________________________________________________________
*Corresponding author: E-mail: renner.nrior1@ust.edu.ng, ogbonna.david@ust.edu.ng;
Current Journal of Applied Science and Technology
40(1): 119-141, 2021; Article no.CJAST.65366
ISSN: 2457-1024
(Past name:
British Journal of Applied Science & Technology,
Past
ISSN: 2231-0843,
NLM ID: 101664541)
Myco-enhanced Bioremediation in Open Field Crude
Oil Contaminated Soil Using Mucor racemosus and
Aspergillus niger
O. Ule
1*
, D. N. Ogbonna
2*
, R. N. Okparanma
3
and R. R. Nrior
2*
1
Institute of Geosciences and Environmental Management, Rivers State University,
Port Harcourt, Nigeria.
2
Department of Microbiology, Faculty of Science, Rivers State University, Port Harcourt, Nigeria.
3
Department of Agricultural and Environmental Engineering, Rivers State University, Port Harcourt,
Nigeria.
Authors’ contributions
This work was carried out in collaboration among all authors. Authors DNO, RNO and RRN designed
the study, performed the statistical analysis, wrote the protocol and wrote the first draft of the
manuscript. Authors RRN, RNO and OU managed the analyses of the study. Authors RNO and OU
managed the literature searches. All authors read and approved the final manuscript.
Article Information
DOI: 10.9734/CJAST/2021/v40i131241
Editor(s):
(1) Dr. Chang-Yu Sun, China University of Petroleum, China.
Reviewers:
(1)
Yemisi Olawore, National mathematical Centre, Nigeria.
(2)
Leili Mohammadi, Zahedan University of Medical Sciences, Iran.
Complete Peer review History:
http://www.sdiarticle4.com/review-history/65366
Received 01 December 2020
Accepted 04 February 2021
Published 02 March 2021
ABSTRACT
Aim:
To assess the Mycoremediation potential of Mucor racemosus and Aspergillus niger in open
field crude oil contaminated soils in Rivers State, Nigeria.
Study Design: The study employs experimental design, statistical analysis of the data and
interpretation.
Place and Duration of Study: Rivers State University demonstration farmland in Nkpolu-
Oroworukwo, Mile 3 Diobu area of Port Harcourt, was used for this study. The piece of land is
situated at Longitude 4°48’18.50” N and Latitude 6
ᵒ
58’39.12” E measuring 5.4864 m x 5.1816 m with
a total area of 28.4283 square meter. Mycoremediation process monitoring lasted for 56 days,
analyses were carried out weekly at 7 days’ interval.
Methodology: Five (5) experimental plots were employed using a Randomized Block Design each
having dimensions of 100 x 50 x 30 cm (Length x Breadth x Height) and were formed and mapped
out on agricultural soil, each plot was contaminated with 22122.25g of Crude Oil except Control 1
Original Research Article
Ule et al.; CJAST, 40(1): 119-141, 2021; Article no.CJAST.65366
120
and left fallow for 6 days after contamination for proper contamination and exposure to natural
environmental factors to mimic crude oil spill site. On the seventh day bio-augmentation process
commenced using two (2) fungal isolates namely Aspergillus niger [Asp] and Mucor rasemosus
[Muc]). Two (2) control plots (P1: Uncontaminated and unamended soil - CTRL 1 US) and P2:
Crude Oil contaminated but unamended soil - CTRL 2 CS); P3 = P5 were contaminated and
amended/bioaugmented (P3: CS+Asp, P4: CS+Muc, P5: CS+Asp+Muc respectively. Soil profile
before and after contamination was assayed while parameters like Temperature, pH, Nitrogen,
Phosphorus, Potassium and Total Petroleum Hydrocarbon (TPH) contents were monitored
throughout the experimental period. Microbial analyses such as Total Heterotrophic Bacteria (THB),
Total Heterotrophic Fungi (THF), Hydrocarbon Utilizing Bacteria (HUB) and Hydrocarbon Utilizing
Fungi (HUF) were recorded. Bioremediation efficiency was estimated from percentage (%) reduction
of Total Petroleum Hydrocarbon (TPH) from day 1 to the residual hydrocarbon at day 56 of bio-
augmentation/ biostimulation plots with the control.
Results: Results revealed actual amount of remediated hydrocarbon and % Bioremediation
Efficiency at 56 days in the different treatment plots (initial TPH contamination value of
8729.00mg/kg) in a decreasing order as follows: CS+Muc (8599.19mg/kg; 33.66%) > CS+Asp+Muc
(8357.31mg/kg; 33.04%) > CS+Asp (8341.58mg/kg; 32.98%) > CTRL 2 -CS (Polluted soil without
amendment) (81.06mg/kg; 0.32%). Microbiological results After fifty-six (56) days of bioremediation
monitoring; %HUB were as follows; CS+Asp+Muc (45.30%) > CS+Asp (40.32%) > CS+Muc
(35.01%) > CTRL 2 –CS (30.43%) > CTRL 1 – US (0%). These results indicate that the presence of
the contaminated crude oil stimulated and sustained the growth of Hydrocarbon Utilizing Bacteria
(HUB) in the contaminated plots (P2 - P3); more so, the higher growth in the enhanced bio-
augmented plots (P3 – P5) shows the positive impact of fungal bio-augmentation in bioremediation
of crude oil polluted soil. It was further observed that treatment plots with higher HUB or HUF had
higher percentage (%) bioremediation efficiency; that is, the higher the sustained HUB and HUF
population, the higher the %Bioremediation process. Hydrocarbon Utilizing Bacteria (Log10 CFU/g):
CS+Asp (4.20) (Day 35) > CS+Muc+Asp (4.18) (Day 35) > CS+Muc (4.08) (Day 28) > CTRL 2 – CS
(3.95) (Day 21) > CTRL 1 – US (3.78) (Day 35). (Fig. 3). Hydrocarbon Utilizing Fungi (Log10
CFU/g): CS+Asp (4.68) (Day 35) > CS+Muc+Asp (4.58) (Day 35) > CS+Muc (4.48) (Day 35) >
CTRL 2 – CS (4.23) (Day 21) > CTRL 1 – US (2.85) (Day 42).
Conclusion: Study showed that bioremediation of crude oil-contaminated soils with Bioaugmenting
fungus singly may be more effective than combination with others depending on the type of
substrate used, nature of the hydrocarbon utilizing organism and environmental conditions prevalent
as seen in Mucor racemosus having higher bioremediation potential than when combined with
Aspergillus niger. Notably, Hydrocarbon Utlilizing Bacteria (HUB) and Hydrocarbon Utilizing Fungi
(HUF) which are the key players in Bioremediation has its peak count value on Day 35, this confers
that nutrient renewal on bioremediation site should be at interval of 35 days for continuous effective
bioremediation of hydrocarbon pollutants. It is therefore recommended that single microbes of high
bioremediation potential could be used since its more effective than consortium of many
hydrocarbon utilizing microbes. Also, nutrient or bio-augmenting microbes’ renewal on
bioremediation site should be at an interval of 35 days for continuous effective bioremediation of
hydrocarbon pollutants.
Keywords: Bioremediation; Bioaugmentation; Mycoremediation; petroleum hydrocarbon; Aspergillus
niger; Mucor racemosus; crude oil contamination; Soils.
1. INTRODUCTION
The release of hydrocarbons into the
environment whether accidentally or due to
human activities is a main cause of water and
soil pollution [1,2]. Soil contamination with
hydrocarbons causes extensive damage of
ecosystems through the food chain
since accumulation of pollutants in animals
and plant tissue may cause death or mutations
[3].
During the past century, industrial production,
urbanization, energy consumption, transportation
and human population have expanded
exponentially, resulting in increased soil, water
and air pollution, which in turn has placed the
environment under substantial pressure [4].
These factors produced a large number of highly
polluted sites all over the planet, usually
containing complex mixtures of toxic and
carcinogenic, organic and inorganic compounds.
Organic contaminants such as total petroleum
Ule et al.; CJAST, 40(1): 119-141, 2021; Article no.CJAST.65366
121
hydrocarbons (TPH) and polycyclic aromatic
hydrocarbons (PAHs) are known mutagens and
carcinogens that enter the food chain together
with lipophilic compounds [5,6,7]. According to
the United States Environmental Protection
Agency the very hazardous chemicals like
benzene, toluene, ethylbenzene, xylenes, and
naphthalene are included in the petroleum
hydrocarbons [8-12]. These pollutants can affect
soil physical characteristics like soil texture and
structural status, compaction, saturated hydraulic
conductivity and penetration resistance [13].
When released on the surface soil, petroleum
hydrocarbons, with specific physico-chemical
characteristics [14] pushes soil toward a
condition undesirable for proper and sustainable
growth of plant and rhizosphere organisms
activity [5,15]. Sources of crude oil/hydrocarbon
release into the environment may include storage
tank leakages. In 2018, storage tanks leakage
and spill accounted for around 116,000 tonnes
discharge of hydrocarbons and crude oil into the
environment. This means that the presence of
these contaminants in soil significantly reduce
the quality of soil and thus minimize the
germinating, growth and health of plants [16].
Therefore, remediation and removing of these
pollutants from soil is necessary for sustainable
environmental health [17,18].
Bioremediation is defined as a process, which
relies on biological mechanisms to reduce
(degrade, detoxify, mineralize or transform)
concentration of pollutants to an innocuous state
[19,20]. The process of pollutant removal
depends primarily on the nature of the pollutant,
which may include: agrochemicals, chlorinated
compounds, dyes, greenhouse gases, heavy
metals, hydrocarbons, nuclear waste, plastics,
and sewage. Apparently, taking into
consideration site of application, bioremediation
techniques can be categorized as ex situ or in
situ. The nature of pollutants, depth and degree
of pollution, type of environment, location, cost
and environmental policies are some of the
selection criteria that are considered when
choosing any bioremediation technique [21].
Petroleum hydrocarbon contamination may occur
through pipelines and oil wells leakages, wrong
methods of disposal of petroleum wastes and
accidental oil spills [22]. The contamination
caused by petroleum hydrocarbon leads to
various carcinogenic and neurotoxic effects.
Therefore, to reduce the hazardous effect of
petroleum hydrocarbon, their control and
treatment strategies through bioremediation are
required [23]. Notably, different oil products like
gasoline diesel or heavy oils can cause soil
contamination [24].
Mycoremediation is defined as a natural or
artificial process in which fungi are used to
degrade contaminants to less toxic or nontoxic
forms, thereby reducing or eliminating
environmental contamination. Ligninolytic fungi
(white rot fungi) can degrade petroleum
hydrocarbons by extracellular lignin modifying
enzymes [25]. These enzymes have very low
substrate specificity, making them suitable for
degradation of a wide range of highly recalcitrant
compounds that is structurally similar to lignin.
The ligninolytic enzymes consist of lignin
peroxidase, manganese peroxidase and laccase
[26]. The spent mushroom compost (SMC)
contains a consortium of hydrocarbon degrading
bacteria and ligninolytic fungi. The SMC contains
large amounts of different types of ligninolytic
enzymes [27]. According to Okerentugba et al
[25] Spent Mushroom Compost can be effective
in the degradation of petroleum hydrocarbon
because of its degrading and ligninolytic
properties. Most studies about hydrocarbon and
petroleum degradation have been conducted on
groundwater aquifers [28] and in laboratory
and/or field studies; however little research has
been carried out on soils. Wegwu et al [29] in
their study indicated that attenuation method is
one of the best techniques for soil refinement in
contaminated soils with crude oil. There are three
methods of attenuation which include; natural
attenuation, biostimulation and bioaugmentation
which are introduced as effective methods for
removal of Total Petroleum Hydrocarbons
(TPHs) from soils [30]. Studies have been
conducted to isolate and characterize
hydrocarbon degraders from oil spill sites but
little have been done to determine the changes in
soil nutrients and TPHs as bioremediation of
the spill site progresses, thus the aim of this
research is to assess the potential of myco-
remediating microbes Mucor and Aspergillus
species in bioremediation of crude oil
contaminated soil and their effects in key soil
nutrient (NPK).
2. MATERIAL AND METHODS
2.1 Description of Area of Study
The area used for this study is a pristine patch of
land within the Rivers State University
Demonstration farmland in Nkpolu-Oroworukwo,
Mile 3 Diobu area of Port Harcourt, Rivers State.
The piece of land is situated at Longitude
4
ᵒ
48’18.50” N and Latitude 6
o
58’39.12” E
Ule et al.; CJAST, 40(1): 119-141, 2021; Article no.CJAST.65366
122
measuring 5.4864m x 5.1816m with a total area
of 28.4283m
2
. This was cleared and sub-
partitioned into 9 blocks of 100cm x 50cm x
30cm giving 214.905 kg of soil in each plot Two
of these plots were designated as pristine and
crude oil polluted soil to serve as controls
respectively (according to method described by
Ogbonna et al [31]. The soil is of sandy clay
texture with specific gravity of 2.57. From these
plots; unpolluted, crude oil polluted and nutrient
amended soil samples were taken for
bioremediation analysis. The study area is shown
in Fig. 1. The choice of the Rivers State
University demonstration farm was premised on
the following factors; enough space, relatively flat
topography, accessibility, availability of water and
secured environment. The site also
demonstrated adequate safeguards for the
protection of human health and the environment.
2.2 Experimental Design
The Randomized Complete Block Design
(RCBD) was used for the study. Each unit of
block or plot measured 100cm x 50cm x 30cm.
The volume of each block gives 214.905 kg
volume of soil taken into consideration the
microbial influence on agricultural soils is in the
range of 0-15cm depth [32].
2.3 Sources of Microbial Isolates
The microorganisms used were fungi specifically
Aspergillus nudilans and Mucor racemosus.
These organisms were isolated from the soil
samples using Sabouroud Dextrose Agar as
selective media for fungi. After which pure
cultures obtained were inoculated onto Modified
Sabouraud Dextrose broth in 500 ml Erlenmeyer
flask loosely plugged with sterile cotton wool for
the growth of the augmenting test organisms.
Broth cultures with an optical density of 0.2 were
used for augmentation.
2.4 Treatment/ Field Application
Five Randomized Complete Block Design
(RCBD) degradative plots according to the
method of Toogood [32] were set-up for the aim
of monitoring bioremediation of crude oil polluted
soil (Table 1). The bioremediation protocol
consists of five RCBD. Two plots of the RCBD
act as control (CTRL 1 for Uncontaminated soil
without treatment while CTRL 2 is for Crude Oil
Contaminated soil without treatment); the other
three plots were treated singly or combined with
bioaugmenting microorganism. They are as
follows.
2.5 Treatment and Application of Crude
Oil and Nutrients
Crude oil used in this experiment was obtained
from AGIP flow station. The stock culture was
prepared by weighing out (PCE analytical
weighing balance PCE-6000), 2122.25g and
dissolve in 1.0 L of distilled water to give initial
crude oil concentration of 2122.25g/l. The soil
was artificially contaminated by spiking the
prepared crude oil concentration on the plots and
allowed to stay for 7 days to ensure volatilization
and sorption of crude oil into the soil matrix
before application of various treatments.
The plots were amended with 750ml of
Aspergillus and 750ml of Mucor accordingly
[31,33]. Plot 1 was uncontaminated (pristine) and
Plot 2 was contaminated but un-amended. These
two plots served as controls. Plots 3-5 were
amended with different concentration of
treatments (Table 1).
2.6 Tilling
The experimental plots were slightly tilled once a
week. This optimizes the transfer of oxygen into
contaminated soils and promotes aerobic
degradation of the organic contaminants.
Table 1. Treatments of Experimental plots using Nutrient amendments and bio-augmenting
organisms
Sample
ID
Plot Code
Crude oil
(g)
Aspergillus(Asp)
(ml)
Mucor (Muc)
(ml)
P1 CTRL 1 (Uncontaminated soil -US) 0 - -
P2 CTRL 2 (Contaminated soil - CS) 2122.25 - -
P3 CS+Asp 2122.25 750 -
P4 CS+Muc 2122.25 - 750
P6 CS+Asp+Muc 2122.25 375 375
P=- Plot; US = Uncontaminated soil; CS = Contaminated soil; Asp = Aspergillus nidulans; Muc = Mucor
racemosus
Ule et al.; CJAST, 40(1): 119-141, 2021; Article no.CJAST.65366
123
2.7 Watering
The plots were watered to 65% water holding
capacity [34] before experimental crude oil
contamination and subsequently at two days’
interval with 600ml of water per plot as required.
2.8 Sample Collection for Analysis
Soil samples for laboratory analysis were
collected on day 1, 7, 14, 21, 28, 35, 42, 49 and
56 in sterile sample container from a depth of 0-
15cm after tilling using soil spatula. Soil samples
collected were made from 4-10 random points
per plots and bulked to form a composite sample.
Small portions (5g) of the composite samples
were collected into sterile bottles using sterile
spatula for microbiological and physicochemical
analysis. All microbiological analysis were carried
out in the Microbiology laboratory of the Rivers
State University within 2 hours after sample
collection while physicochemical analysis was
carried out at Pollution Control and
Environmental Management (POCEMA) and
Giolee Global Resources Laboratories both in
Port Harcourt, Rivers State. Soil samples were
stored at 14±2
o
C for future analysis [34].
2.9 Microbiological Analysis of Soil
Samples
The following Media were used for microbial
enumeration and isolation
2.9.1 Nutrient agar
Nutrient agar (NA) was used as a general-
purpose medium because it supports the growth
of a wide range of non-fastidious
microorganisms. Nutrient agar of Becton
Dickson and Company, USA was used for the
isolation of Total Heterotrophic Bacteria (THB) by
preparing/weighing out (with a normal calibration)
28 grams of the Nutrient agar into 1000ml of
distilled water and then sterilized/autoclaved at
121
0
C for 15 minutes according to the
manufacturer specification
2.9.2 Oil agar medium
Oil agar medium was prepared for the isolation of
hydrocarbon utilizing bacteria. Oil-agar medium
was prepared by the method of Modified Salts
Medium (MSM) of Nrior and Odokuma [34]; Nrior
and Echezolom [35]. The medium was prepared
with a composition of K
2
HPO
4
(0.5g),
MgSO
4
.7H
2
O (0.3g), NaCl
2
(0.3g), MnSO
4
.H
2
O
(0.2g), FeSO
4
.6H
2
O (0.02g), NaNO
3
(0.03g),
ZnCl
2
(0.3g) and agar (15g) into 1litre of distilled
water. 1% of pure Bonny light crude oil was
added to the mixture and then autoclaved at
121
°
C for 15 minutes. The medium was used for
the isolation, enumeration and preliminary
identification of petroleum utilizing bacteria (oil
degraders). The medium was then prepared by
the addition of 1% (v/v) crude oil sterilized with
0.22Millipore filter paper to sterile MSM cooled to
45
o
C under aseptic condition. The MSM and
crude oil were then mixed thoroughly and
dispensed into sterile Petri dishes to set.
2.9.3 Sabouroud dextrose agar
Sabouroud Dextrose Agar (SDA) was used for
the isolation of fungi isolates. Media was
prepared by weighing out 65g into 100ml of
distilled water and using the manufacturer’s
specification, depending on the number of plates
used. After the preparation it was autoclaved at
121
0
C for 15 minutes and then the media was
aseptically poured into plates for inoculation.
2.10 Glassware and Media Sterilization
The glassware used for the laboratory analysis
were sterilized in a hot air oven at 160
o
C for 1-
3hours. The sterilization for the media and water
used for the serial dilutions were carried out in an
autoclave at 120
o
C and 15 pounds per square
inch (psi) for 15 minutes while sugars for
fermentation and metabolism tests were
sterilized in the autoclave for 5-10 minutes.
2.11 Microbiological Analyses
2.11.1 Microbial estimation
The total heterotrophic bacteria (THB), the
hydrocarbon utilizing bacteria (HUB), total
heterotrophic fungi (THF) and hydrocarbon
utilizing fungi (HUF) were determined using the
spread plate count method on nutrient agar
according to APHA [36] as cited by Chikere et
al., [37]; Oliveira et al., [38] and Nrior and Mene
[2].
2.11.2 Enumeration and Isolation of pure
culture
Colonies and spores that grew on NA and SDA
from the baseline and bioremediation set-up after
incubation were enumerated. Similarly, colonies
and spores were picked for subculture to get
pure cultures and so were those that grew on
MSA plates. Pure culture of fungi were stored on
SDA slants, while those of bacteria isolates were
stored in 10% glycerol, all in Bijou bottles.
Ule et al.; CJAST, 40(1): 119-141, 2021; Article no.CJAST.65366
124
2.11.3 Identification of fungal isolates
Two fungal spores that utilized petroleum
hydrocarbons as their sole carbon energy source
were viewed macroscopically and
microscopically (using Lactophenol Cotton Blue
Stain and the slide culture technique).
2.11.4 Wet mount preparation
A flamed needle was used to pick spores with
mycelium from SDA plate and aseptically placed
onto two drops of Lactophenol Cotton Blue
(LPCB) on a grease-free slide. The spores were
thinned out to enable easy identification. A cover
slip was placed on the slide and the stained fungi
viewed using X40 magnification [39] and other
microscopic and cultural characteristics were
further used in the identification of the fungal
isolates of the bioremediation set up [40].
2.11.5 Slide culture method
From the sterile SDA, a small square shaped
piece was cut and placed to fit onto a grease-free
slide under a cover slip. Using a flamed needle, a
growing fungal spore was picked from SDA plate
and embedded into the four sides of the piece of
agar and a cover slip placed on top of the
embedded piece of agar. Moistened filter paper
was placed in a petri-dish under the glass slide.
The petri-dish was covered and incubated at
37
o
C until sporulation occurred [39].
2.11.6 Purification and Preservation of Pure
Cultures
The Fungal isolates were inoculated onto
Sabouraud Dextrose Broths in 500ml Erlenmeyer
flask loosely plugged with sterile cotton wool
respectively. The broth cultures were incubated
for 5 days at 28
o
C. Serial dilution was made to
determine the number of cells per 0.1 ml aliquot.
2.11.7 Enumeration and Isolation of pure
culture
Colonies and spores that grew on NA and SDA
from the baseline and bioremediation set-up after
incubation were enumerated. Similarly, colonies
and spores were picked for subculture to get
pure cultures and were those that grew on MSA
plates. Pure culture of fungi was stored on SDA
slants, while those of bacteria isolates were
stored in 10% glycerol, all in Bijou bottles.
The colonies counted were expressed as Colony
Forming Unit (CFU) per gram of soil using the
formula:
T =
Where
T = total number of colonies in cfu/g soil
N = number of colonies counted on the plate
V = volume of inoculum plated i.e. 0.1ml
DF = dilution factor used for plating (10
6
)
Total Heterotrophic Bacterial count =
2.11.8 Bioremediation evaluation procedure
All plots were tilled twice weekly to ensure proper
aeration and even distribution of crude oil and
bioaugmenting agents/microbes. Samples were
taken at regular interval of days 1, 7, 14, 21, 28,
35, 42, 49 and 56 for microbiological and
selected physicochemical analyses.
2.9 Bioremediation Analysis
2.9.1 Percentage (%) bioremediation analysis
The method of Nrior and Mene [2] was used in
calculating the percentage of Bioremediation in
the experiment. The process followed the steps
stated.
Step i: The amount of pollutant remediated
equals to Initial Concentration of pollutant
(Week 1) minus the Final Concentration of
pollutant at the end of experiment (Last day or
Week 8)
Bc = Ic – Fc same as ARx = ICx – FCx
Where:
Bc (ARx) = Amount of pollutant remediated in
plot x
Ic (ICx) = Initial Concentration of pollutant in
plot x (week 1)
Fc (FCx) = Final Concentration of pollutant in
plot x (week 8)
Step ii: The percentage (%) Bioremediation
equals Amount of pollutant divided by the Initial
Concentration of pollutant (week 1), multiplied by
100
% Bioremediation = (Bc/Ic) x 100 same as %
Bioremediation = (ARx/ICx) x 100 (Nrior and
Mene), [2]
Ule et al.; CJAST, 40(1): 119-141, 2021; Article no.CJAST.65366
125
2.9.2 Actual %bioremediation
Step 1: Calculate Amount of Hydrocarbon (Crude
oil) remediated in Control without experimental
Contamination (CTRL 1 – US) [Note: This is
essential where there are heavy activities of oil
companies, exploration, spillage, marketing of
crude oil etc, that makes almost impossible for
any piece of land to be completely free from
residual crude oil contamination; example, Niger
Delta Region of Nigeria]
For Control without experimental
contamination CTRL 1 – US
AR
CTRL1-US
= IC
CTRL1-US
- FC
CTRL1-US
Step 2: Actual Amount Remediated (AAR) equals
Amount Remediated in each Treatment (ARx)
minus Amount Remediated in Control (ARc)
(Experimental Uncontaminated soil CTRL 1 - US)
AAR = ARx – ARc
%AAR = (ARx/∑ARx) *100
2.9.3 Physicochemical analysis of selected
parameters
The Physicochemical property of the soil sample
was determined before experimental
contamination/pollution of the soil to establish the
baseline parameters and subsequently after
crude oil contamination and nutrient application
for the duration of bioremediation process for
selected parameters. The following selected
parameters including; soil texture, particulate
size, moisture content, pH, temperature,
phosphate, nitrate (NO
3
-
), sulphate, total organic
carbon, electrical conductivity, and moisture
content were determined using the methods from
APHA [36]. Soil texture was determined using
sieves of different sizes – Master Sizer 2000
(Malner International), while moisture content
was determined by drying 10g of the soil sample
in an oven at 80
o
C. Then 10g of oven dried soil
was placed on filter papers (Whatman No. 42)
and filtered into Buchner funnels. De-ionized
water was added slowly until the water level was
just above the soil surface, then saturated and
dipped into the flask. The funnel was removed
and left to dry overnight. The soil was left for
24hrs, rewetted and the whole apparatus
reweighed. The percentage moisture content of
the soil in triplicate was then determined and
calculated as water holding capacity (100%).
Soil pH was determined using a pH meter (pH-
911 Pen type). The temperature of the soil was
determined using a mercury thermometer, by
inserting the thermometer into the tilled soil for a
period of 3-5 minutes and taking the reading
immediately the thermometer is removed from
the soil.
2.9.4 Total petroleum hydrocarbon (TPH)
Residual Total Petroleum Hydrocarbons (TPH)
was extracted from the soil samples and
quantified using Gas Chromatograph – Flame
Ionization Detector (GC-FID) Agilent 7890A,
according to the methods of ASTDM 3921 and
US EPA 8015 analytical protocol (TPI, 2007) as
reported by Chikere et al. [37] and in accordance
with Nigerian requirements of Department of
Petroleum Resources (DPR), National Oil Spill
Detection Response Agency (NOSDRA) and
Federal Ministry of Environment (FMEnv).
Samples were collected in a sealed sample
container from Giolee Global Resources
laboratory. Samples were kept in a cooler with
icepack at 4°c, labeled appropriately and sent to
the laboratory for analysis. All samples were
analyzed in duplicates while ensuring precision
and reliability of results through standard quality
assurance and control procedures.
2.9.5 Determination of nitrate (NO
3
2-
) in soil
sample
5g of soil sample was weighed into a shaking
bottle.125ml of distilled water was added and
shaken for 10minutes on a rotary shaker and
then filtered to obtain the extract. 1ml of the
extract was transferred into 10ml volumetric
flask. 0.5ml of Brucine reagent was then added.
2ml of conc. sulphuric acid was rapidly added
and mixed for about 30seconds. The flask was
allowed to stand for 5minutes; 2ml of distilled
water was added and mixed for about
30seconds. Flask was allowed to stand in cold
water for about 15minutes.The absorbance was
measured at wavelength of 470nm.
2.9.6 Determination of phosphate (PO
4
3-
) in
soil sample
25ml of 2.5% Acetic acid was added to 1g of soil
sample and shaken for 30minutes. The
suspension was filtered through a filter paper.
10ml of the extract was transferred into 50ml
volumetric flask. Extract was diluted with distilled
water until the flask is about 2/3 full. 2ml of
Ammonium Molybdate reagent was added and
mixed with extract. 2ml of stannous chloride was
also added and mixed; the solution was diluted to
50 ml mark with distilled water. The flask was
Ule et al.; CJAST, 40(1): 119-141, 2021; Article no.CJAST.65366
126
allowed to stand for 30minutes, and the
absorbance was measured at wavelength of 690
nm.
2.9.7 Determination of sulphate (SO
4
2-
) in soil
sample
25ml of the extracting solution was added to 5g
of soil sample and shaken for 30minutes and the
suspension was filtered through a filter paper.
5ml of the extract was transferred into 50ml
volumetric flask. 5ml of 50% acetic acid was
added and 1ml of H
3
PO
4
was added and mixed.
The solution was diluted with distilled water to
about ¾ of the flask. 1g of Barium chloride was
added and mixed. The solution was left to stand
for 10 times, then 1ml of 0.5% gum acacia was
added to the solution and made up to 50ml with
distilled water, and the absorbance was
measured at 425nm.
2.9.8 Statistical analysis
Data obtained from the bioremediation set up
were subjected to statistical analysis using
computer based program, SPSS version 22 for
Analysis of Variance (ANOVA) and Excel on
microbiological, Total petroleum hydrocarbons
and physicochemical parameters to compare
data between soils in all treatments and controls
and test whether the different nutrient
amendments given to the crude oil contaminated
soils were statistically significant at a confidence
level of 95% or P>0.05.The results expressed as
Mean±SD and regression analysis.
3. RESULTS AND DISCUSSION
3.1 Microbial and Physico-chemical
Properties of the Soil Prior to
Application of Various Treatments for
Bioremediation Evaluation
Baseline Physico-chemical and Morphological
properties of the soil prior to Bioremediation.
Table 2 shows the baseline physico-chemical
and microbiological properties of the soil before
the application of various bioremediation
treatment approaches. Notably, key parameters
determined were pH, electrical conductivity,
Nitrate, potassium, phosphorus, sulphate,
phosphate, moisture content, total organic
carbon and particle size. The microbial analysis
were Total Heterotrophic Bacteria (THB), Total
Heterotrophic Fungi (THF), Hydrocarbon Utilizing
Bacteria (HUB) and Hydrocarbon Utilizing Fungi
(HUF) while the concentration of total petroleum
hydrocarbon (TPH) was also determined. The
baseline results revealed that the pH was 7.01
for uncontaminated soil and 6.80 for
contaminated soil. The electrical conductivity was
500µS/cm for uncontaminated soil and
590µS/cm for contaminated soil. TPH value was
as low as 87.89mg/kg in the uncontaminated soil
and 8729mg/kg in the contaminated soil.
Table 2. Baseline Physico-chemical and Microbiological properties of the soil prior to
application of various treatments for Bioremediation evaluation
S/N
Parameter unit Uncontaminated soil Contaminated soil
1 pH - 7.01 6.80
2 Temperature
°
C 26.78 28.56
1 Electrical Conductivity µS/CM
500.00 590.00
2 Nitrate mg/kg 506.95 454.72
3
Potassium, K
mg/kg
3.01
1.85
4 Phosphorus,P mg/kg 2.49 2.14
5 Sulphate SO
4
2-
mg/kg 0.026433 0.020025
6
Phosphate PO
4
3-
mg/kg
0.00156
0.00167
7 Moisture Content % 15.95 18.67
8 Total Organic carbon (TOC) % 0.88 0.28
9 Particle size (>75µm) % 81.10 50.90
10 Total Petroleum Hydrocarbon (TPH) mg/kg 87.89 8729
11 Total Heterotrophic Bacteria (THB) CFU/g 5.0 x 10
8
2.3 x 10
8
12 Total Heterotrophic Fungi (THF) CFU/g 8.0 x 10
3
1.4 x 10
4
13 Hydrocarbon Utilizing Bacteria (HUB) CFU/g 0 3.0 x 10
4
14 Hydrocarbon Utilizing Fungi (HUF) CFU/g 3.0 x 10
3
9.0 x 10
4
Ule et al.; CJAST, 40(1): 119-141, 2021; Article no.CJAST.65366
127
Soil physical properties define movement of air
and water/dissolved chemicals through soil, as
well as conditions affecting germination, root
growth, and erosion processes. Soil physical
properties form the foundation of several
chemical and biological processes. The physical,
chemical, and biological properties collectively
determine the quality of the soil [41]. The soil's
chemical properties are inherited from the
processes of soil formation, during weathering
and transport of the parent material from which
the soil has formed. Thus the chemical nature of
the rocks and minerals and the intensity of the
weathering processes are fundamental in
determining the chemical properties of the soil
[42].
In soil, electrical conductivity (EC) is a measure
of the ability of the soil to conduct an electrical
current. Most importantly to fertility, EC is an
indication of the availability of nutrients in the
soil. The higher the EC, the more negatively
charged sites (clay and organic particles) there
must be in the soil, and therefore the more
cations (which have a positive charge) there are
that are being held in the soil. Sodium (Na
+
),
ammonium (NH
4
+
), potassium (K
+
), calcium
(Ca
2+
), magnesium (Mg
2+
), hydrogen (H
+
), iron
(Fe
2+
), aluminum (Al
3+
), copper (Cu
2+
), zinc (Zn
2+
)
and manganese (Mn
2+
) are some examples of
these cations that are beneficial to plants. As
with most things in the soil, it is important that the
EC does not get too high either, as too many of
these nutrients, especially Na and Mg, can be
detrimental to soil health. Optimal EC levels in
the soil therefore range from 110-570 milli
Siemens per meter (mS/m). Too low EC levels
indicate low available nutrients, and too high EC
levels indicate an excess of nutrients. Low EC’s
are often found in sandy soils with low organic
matter levels, whereas high EC levels are usually
found in soils with high clay content [43].
3.2 Microbiological Evaluation during
Bioremediation of Crude Oil Polluted
Soil
The bacteria genera isolated form crude oil
polluted soil were: Bacillus, Micrococcus,
Comamonas, Klebsiella, Chryseobactrium,
Pseudomonas, Pseudomona, Staphylococcus
and Nitrosomonas while fungal isolates were:
Aspergillus sp., Penicillium sp., Cladosporium
sp., Mucor sp., Microsporium sp.
The results of the microbial evaluation of the
study are shown in Fig. 2-5. Counts for Total
Heterotrophic Bacteria (THB), Total
Heterotrophic Fungi (THF), Hydrocarbon Utilizing
Bacteria (HUB) and Hydrocarbon Utilizing Fungi
(HUF) during bioremediation of crude oil polluted
soil were all determined in this study. Significant
microbial counts for Total Heterotrophic Bacteria
(Log10 CFU/g) were recorded on day 42, 49 and
56 of the bioremediation; The highest count for
each plots were as follows; CTRL 2 – CS (9.86)
(Day 56) > CTRL 1 – US (9.24) (Day 49) >
CS+Muc (9.12) (Day 49) > CS+Muc+Asp (9.03)
(Day 42) = CS+Asp (9.03) (Day 28) (Fig. 1).
Generally, there seems to be peak count on day
49 and a decline in the THB count on Day 56.
Total Heterotrophic Fungi (Log10 CFU/g): CTRL
2 –CS (5.20) (Day 28) > CS+Muc+Asp (4.95)
(Day 56) > CS+Muc (4.93) (Day 35) > CS+Asp
(4.78) (Day 42) > CTRL 1 – US (3.95) (Day 48).
(Fig. 2). Hydrocarbon Utilizing Bacteria (Log10
CFU/g): CS+Asp (4.20) (Day 35) > CS+Muc+Asp
(4.18) (Day 35) > CS+Muc (4.08) (Day 28) >
CTRL 2 – CS (3.95) (Day 21) > CTRL 1 – US
(3.78) (Day 35). (Fig. 3). Hydrocarbon Utilizing
Fungi (Log10 CFU/g): CS+Asp (4.68) (Day 35) >
CS+Muc+Asp (4.58) (Day 35) > CS+Muc (4.48)
(Day 35) > CTRL 2 – CS (4.23) (Day 21) > CTRL
1 – US (2.85) (Day 42). (Fig. 4) Notably,
Hydrocarbon Utlilizing Bacteria (HUB) and
Hydrocarbon Utilizing Fungi (HUF) which are the
key players in Bioremediation has its peak count
value on Day 35, this confers that nutrient
renewal on Bioremediation site should be at
interval of 35 days for continuous effective
bioremediation of hydrocarbon pollutants.
Evaluation of Percentage (%) Hydrocarbon
Utilizers during enhanced Bioremediation of
Crude Oil Contaminated soil showed Significant
growth in plots contaminated with crude oil while
at day 56 the Uncontaminated plot used as
Control 1 recorded zero percent (Table 3). The
0% HUB and HUF on Day 56 is a clear indication
of the absence of Crude oil as their carbon
source.
After fifty six (56) days of bioremediation
monitoring; %HUB were as follows;
CS+Asp+Muc (45.30%) > CS+Asp (40.32%) >
CS+Muc (35.01%) > CTRL 2 –CS (30.43%) >
CTRL 1 – US (0%), the mean value has same
trend (Table 3-4). This result indicates that the
presence of the contaminated Crude Oil
stimulated and sustained the growth of
Hydrocarbon Utilizing Bacteria (HUB) in the
contaminated plots (P2 - P3); more so, the higher
growth in the enhanced bio-augmented plots (P3
Ule et al.; CJAST, 40(1): 119-141, 2021; Article no.CJAST.65366
128
– P5) shows the positive impact Myco (fungal)
bio-augmentation in bioremediation of Crude oil
polluted soil. It was further discovered that
treatment plots with higher HUB or HUF had
higher percentage (%) bioremediation; that is,
the higher the sustained HUB and HUF
population, the higher the %Bioremediation.
(Table 3-4, Fig 3-5).
Assessment of Hydrocarbon Utilizing Fungi
showed similar trend to that of HUB, with the
enhanced treatment plots (Mycobio-augmented
plots P3 – P5)) having higher counts, though
CS+Muc (91.87%) (Day 56) with Net %HUF
(12.18%) > CS+Asp+Muc (88.32%) (Net %HUF
12.03%)
Biodegradation mediated by indigenous microbial
communities is a key process by which
petroleum hydrocarbons are mineralized and
removed from contaminated environments. Thus,
microbial oil biodegradation is recognized as one
of the most important methods for petroleum
hydrocarbon remediation. Most petroleum
hydrocarbons are biodegradable under aerobic
conditions. Hydrocarbon-oxidizing bacteria
capable of growth on aliphatic and aromatic
hydrocarbons are found in many genera. In the
presence of O
2
, the initial steps in the bacterial
degradation of hydrocarbons rely on
oxygenases. These oxygenases are membrane-
bound, the cell must come into direct contact with
their water-insoluble substrates. The oxygenases
are group-specific for example, therefore some
degrade specific fractions of alkanes, whereas
others work on aromatics or cyclic hydrocarbons,
it follows that only a mixture of different
microorganisms can efficiently degrade crude oil
and petroleum fractions [2,43]
3.3 Physico-Chemical Properties of Soil
during Bioremediation
The physico-chemical characteristics of the bio-
remediated soil was duly conducted. This was
done by determining the pH, temperature,
nitrogen, phosphorus and potassium
concentrations. The pH ranged between 5.68 –
7.19 with its mean peak value 6.95±0.20
recorded in the First Control: Uncontaminated
soil without Bio-amendment (CTRL 1 – US) plot
(Fig. 6, Table 5). The Crude Oil contaminated
plots had relatively lower pH; this implies that
crude oil had a reductive effect on the soil pH
tending toward acidity. Temperature also ranged
between 27.62±0.81 - 28.77±0.96
o
C with its peak
in the Second Control plot - Crude Oil
Contaminated soil without Bio-amendment
(CTRL 2 - CS). Temperature range were
relatively same between the bioremediation
group and the control group but were higher in
the Crude Oil Contaminated plot than the
Uncontaminated Control plots (Fig. 7, Table 5).
Two things seemed clear; that the presence of
Crude oil in soil tends to lower soil pH and
increase its Temperature.
Nitrogen value in the experimental plots ranged
from 344.32 – 549.22mg/kg with its mean peak
value as 494.39±24.14mg/kg. Similar trends
were observed with phosphorus and potassium
(Table 5-6, Fig. 8-10). Noteworthy, the control
groups varied significantly from the CS+Asp,
CS+Muc and CS+Asp+Muc as featured in Fig.
10, for Potassium day 14 (CS+Asp+Muc) and
(CS+Muc); while Total Petroleum Hydrocarbon
(TPH) (mg/kg) in Fig. 11 and 12, Day 1-56 for
Control 2 (Crude Oil Contaminated soil without
amendment CTRL 2- CS) varied significantly
from the Contaminated and amended plots
(CS+Asp, CS+Muc and CS+Asp+Muc). In a
study on the effects of organic manures on the
physico-chemical properties of crude oil polluted
soils, the percentage pH, percentage total
nitrogen, phosphorus and exchangeable bases
(Ca, K and Mg) significantly decreased along
with a decrease in the hydrocarbon content of
the soil in that study [27,44]. Elsewhere, a study
on the physicochemical properties of crude oil
contaminated soils as influenced by cow dung
and showed that the percentage of Nitrogen,
Phosphorus, Potassium and pH significantly
decreased two weeks after crude oil
contamination, thereby suggesting that the
addition of crude oil may have adverse effect on
the physicochemical properties of soil [31]. The
physicochemical parameters of the
bioremediation study of a contaminated soil
resulted in a decrease of the total organic carbon
(56.64 %), sulfate (57.66 %), nitrate (57.69 %),
phosphate (57.73 %), sodium (57.69 %),
potassium (57.68 %), calcium (57.69 %) and
magnesium (57.68 %) except pH (3.90 %) that
slightly increased [45].
As depicted in Table 7 and Fig. 12-13, the total
petroleum hydrocarbon concentration in the
contaminated soil CS ranged between 8562.46 -
8729.00 mg/kg with the peak concentration being
recorded in day 1 and a very slight negligible
decline between day 7 and 56. Notably, no
particular trend of decline was observed. TPH
levels during bioremediation showed that a
progressive decline in the concentration was
observed from day 7 to day
56 with the highest
decline being recorded at the end of the
bioremediation at day 56. While the values in day
1 was 87.89mg/kg for the uncontaminated soil
used as control, the 8729.00 mg/kg, the value at
day 56 for all bioremediation option had a range
of 2.41 -
779.99 mg/kg. The least TPH level at
day 56 was recorded in CS+Muc and
CS+Asp+Muc+SMS with values of 129.81 mg/kg
and 258.40 mg/kg respectively.
The findings of the present study conforms with
the findings of a study by Benyahia & Embaby
[46] wh
o reported a total petroleum hydrocarbon
(TPH) reduction of 77% over 156 days longer
than the bioremediation period in the present
study. In another related study, Ebuehi et al. [47]
reported TPH concentration of 1.1004 x10
mg/kg of the sandy soil was ach
spiking and tilling. In this same study, there was
a reduction in the TPH level from 300mg/kg after
8 weeks, to 282mg/kg after 10 weeks.
Typically, Petroleum hydrocarbons are complex
substances formed from hydrogen and carbon
molecules and so
metimes containing other
impurities such as oxygen, sulfur, and nitrogen.
They are highly lipophilic and unless they are of
high viscosity (e.g., tar and motor oil), they are
generally readily absorbed through skin and
intact mucosae [43]. TPH is a mixture
Fig. 1. Total
Heterotrophic Bacteria (THB
Ule et al.; CJAST, 40(1): 119-141, 2021
; Article no.CJAST.65366
129
56 with the highest
decline being recorded at the end of the
bioremediation at day 56. While the values in day
1 was 87.89mg/kg for the uncontaminated soil
used as control, the 8729.00 mg/kg, the value at
day 56 for all bioremediation option had a range
779.99 mg/kg. The least TPH level at
day 56 was recorded in CS+Muc and
CS+Asp+Muc+SMS with values of 129.81 mg/kg
The findings of the present study conforms with
the findings of a study by Benyahia & Embaby
o reported a total petroleum hydrocarbon
(TPH) reduction of 77% over 156 days longer
than the bioremediation period in the present
study. In another related study, Ebuehi et al. [47]
reported TPH concentration of 1.1004 x10
4
mg/kg of the sandy soil was ach
ieved after
spiking and tilling. In this same study, there was
a reduction in the TPH level from 300mg/kg after
8 weeks, to 282mg/kg after 10 weeks.
Typically, Petroleum hydrocarbons are complex
substances formed from hydrogen and carbon
metimes containing other
impurities such as oxygen, sulfur, and nitrogen.
They are highly lipophilic and unless they are of
high viscosity (e.g., tar and motor oil), they are
generally readily absorbed through skin and
intact mucosae [43]. TPH is a mixture
of
chemicals, but they are all made mainly from
hydrogen and carbon, called hydrocarbons.
Scientists divide TPH into groups of petroleum
hydrocarbons that act alike in soil or water.
These groups are called petroleum hydrocarbon
fractions. Also, PAHs are
constituents of
petroleum hydrocarbons that have become
ubiquitous in the environment because of the
persistent exploitation of crude oil and its
derivatives. Such pollutants may undergo
photolysis, chemical oxidation, volatilization,
leaching, bioaccumula
tion, and/or adsorption in
soil. The degradation of these PAHs by the
bioremediation process was achieved via aerobic
process [48].
Actual Amount of remediated hydrocarbon and %
Bioremediation Efficiency at 56 days in the
different treatment plots (initi
contamination value of 8729.00mg/kg) in a
decreasing order as follows: CS+Muc
(8599.19mg/kg; 33.66%) > CS+Asp+Muc
(8357.31mg/kg; 33.04%) > CS+Asp
(8341.58mg/kg; 32.98%) > CTRL 2
soil without amendment) (81.06mg/kg; 0.32%).
Microbiologi
cal results After fifty six (56) days of
bioremediation monitoring; %HUB were as
follows; CS+Asp+Muc (45.30%) > CS+Asp
(40.32%) > CS+Muc (35.01%) > CTRL 2
(30.43%) > CTRL 1 –
US (0%) (Table 7 and
Fig. 13).
Heterotrophic Bacteria (THB
–
Log10 CFU/g) during enhanced bioremediation of
Crude Oil contaminated soil
; Article no.CJAST.65366
chemicals, but they are all made mainly from
hydrogen and carbon, called hydrocarbons.
Scientists divide TPH into groups of petroleum
hydrocarbons that act alike in soil or water.
These groups are called petroleum hydrocarbon
constituents of
petroleum hydrocarbons that have become
ubiquitous in the environment because of the
persistent exploitation of crude oil and its
derivatives. Such pollutants may undergo
photolysis, chemical oxidation, volatilization,
tion, and/or adsorption in
soil. The degradation of these PAHs by the
bioremediation process was achieved via aerobic
Actual Amount of remediated hydrocarbon and %
Bioremediation Efficiency at 56 days in the
different treatment plots (initi
al TPH
contamination value of 8729.00mg/kg) in a
decreasing order as follows: CS+Muc
(8599.19mg/kg; 33.66%) > CS+Asp+Muc
(8357.31mg/kg; 33.04%) > CS+Asp
(8341.58mg/kg; 32.98%) > CTRL 2
-CS (Polluted
soil without amendment) (81.06mg/kg; 0.32%).
cal results After fifty six (56) days of
bioremediation monitoring; %HUB were as
follows; CS+Asp+Muc (45.30%) > CS+Asp
(40.32%) > CS+Muc (35.01%) > CTRL 2
–CS
US (0%) (Table 7 and
Log10 CFU/g) during enhanced bioremediation of
Fig. 2. Total Heterotrophic Fungi (THF
Fig. 3. Hydrocarbon Utilizin
g Bacteria (HUB
of Crude Oil contaminated soil
Ule et al.; CJAST, 40(1): 119-141, 2021
; Article no.CJAST.65366
130
Fig. 2. Total Heterotrophic Fungi (THF
–
Log10 CFU/g) during enhanced bioremediation of
Crude Oil contaminated soil
g Bacteria (HUB
–
Log10 CFU/g) during enhanced bioremediation
of Crude Oil contaminated soil
; Article no.CJAST.65366
Log10 CFU/g) during enhanced bioremediation of
Log10 CFU/g) during enhanced bioremediation
Fig. 4. Hydrocarbon Utilizing Fungi (HUF
Fig. 5. Net percentage of hydrocarbon
Ule et al.; CJAST, 40(1): 119-141, 2021
; Article no.CJAST.65366
131
Fig. 4. Hydrocarbon Utilizing Fungi (HUF
–
Log10 CFU/g) during enhanced bioremediation of
Crude Oil contaminated soil
Fig. 5. Net percentage of hydrocarbon
utilizers
; Article no.CJAST.65366
Log10 CFU/g) during enhanced bioremediation of
Ule et al.; CJAST, 40(1): 119-141, 2021; Article no.CJAST.65366
132
Table 3. Percentage (%) Hydrocarbon utilizers during enhanced Bioremediation of crude oil contaminated soil
%HUB
Experimental plot
Day 1
Day 7
Day 14
Day 21
Day 28
Day 35
Day 42
Day 49
Day 56
%HUB P1 CTRL 1 - US 0 0 32.54 36.11 39.65 41.72 38.41 0.00 0.00
P2 CTRL 2 - CS 41.63 37.80 33.67 44.18 42.14 44.38 41.95 38.33 30.43
P3 CS+Asp 36.23 34.84 33.90 45.38 44.30 46.82 43.94 42.73 40.32
P4 CS+Muc 39.05 41.04 38.54 44.25 44.93 44.20 42.81 40.57 35.01
P5 CS+Asp+Muc 0 0 33.94 45.92 44.25 46.50 45.18 48.95 45.30
%HUF P1 CTRL 1 - US 63.59 73.54 74.03 75.14 69.23 71.43 73.08 58.23 0
P2 CTRL 2 - CS 95.18 78.46 103.29 107.09 99.04 102.75 100 92.31 77.92
P3 CS+Asp 97.47 80.61 96.51 95.63 93.43 98.53 94.35 86.71 88.32
P4 CS+Muc 92.5 94.89 99.29 98.44 96.12 90.87 89.39 84.03 91.67
P5 CS+Asp+Muc 91.13 87.84 100 96.41 95.87 95.82 100.67 96.63 74.75
Table 4. Mean Standard Deviation and Percentage Microbial (Log10 cfu/g) counts during Bioremediation of Crude Oil Contaminated Soils+
Microbial populations (log
10
cfu/g)
Plot
Treatments
THB
THF
HUB
HUF
%HUB
%HUF
Net %HUB
Net %HUF
P1 CTRL 1 - US 9.06±0.16
b
3.83±0.09
b
1.91±1.82
a
3.27±1.24
a
20.94±20.02
a
62.02±23.91
a
5.54 9.48
P2 CTRL 2 - CS 8.97±0.40
a
4.12±0.42
a
2.86±1.63
a
3.90±0.38
a
32.27±4.79
a
95.12±10.54
a
8.29 11.31
P3 CS+Asp 8.82±0.26
ab
4.56±0.31
b
3.62±0.50
a
4.20±0.31
a
40.94±4.84
a
92.40±5.95
ab
10.50 12.18
P4 CS+Muc 8.88±0.29
a
4.47±0.35
a
3.33±1.28
a
4.15±0.30
ab
41.16±3.27
a
93.02±4.78
b
9.65 12.03
P5 CS+Asp+Mc 8.76±0.33
ab
4.53±0.31
a
3.03±1.75
a
4.22±0.34
a
34.45±19.97
a
93.24±8.00
a
8.79 12.24
**means with the same superscript along the columns are not significantly different (p>0.05)
THB = Total Heterotrophic Bacteria, THF = Total Heterotrophic Fungi, HUB = Hydrocarbon Utilizing Bacteria, HUF = Hydrocarbon Utilizing Fungi, P=- Plot; US =
Uncontaminates soil; CS = Contaminated soil; Asp = Aspergillus niger; Muc = Mucor racemosus
Fig. 6. Variation in pH during bioremediation of crude
Fig. 7. Variation in temperature (
Ule et al.; CJAST, 40(1): 119-141, 2021
; Article no.CJAST.65366
133
Fig. 6. Variation in pH during bioremediation of crude oil contaminated soil
Fig. 7. Variation in temperature (
°
C) during bioremediation of crude oil contaminated Soil
; Article no.CJAST.65366
oil contaminated soil
C) during bioremediation of crude oil contaminated Soil
Fig. 8. Variation in nitrogen (mg/kg) during bioremediation of crude oil contaminated soil
Fig. 9. Variation in phosphorus (mg/kg) d
Ule et al.; CJAST, 40(1): 119-141, 2021
; Article no.CJAST.65366
134
Fig. 8. Variation in nitrogen (mg/kg) during bioremediation of crude oil contaminated soil
Fig. 9. Variation in phosphorus (mg/kg) during bioremediation of crude oil contaminated soil
; Article no.CJAST.65366
Fig. 8. Variation in nitrogen (mg/kg) during bioremediation of crude oil contaminated soil
uring bioremediation of crude oil contaminated soil
Fig. 10. Variation in potassium (mg/kg) during bioremediation of crude oil contaminated soil
Fig. 11. Variation in total petroleum hydrocarbon (tph) (mg/kg) during bioremediation of cr
Ule et al.; CJAST, 40(1): 119-141, 2021
; Article no.CJAST.65366
135
Fig. 10. Variation in potassium (mg/kg) during bioremediation of crude oil contaminated soil
Fig. 11. Variation in total petroleum hydrocarbon (tph) (mg/kg) during bioremediation of cr
oil contaminated soil
; Article no.CJAST.65366
Fig. 10. Variation in potassium (mg/kg) during bioremediation of crude oil contaminated soil
Fig. 11. Variation in total petroleum hydrocarbon (tph) (mg/kg) during bioremediation of cr
ude
Ule et al.; CJAST, 40(1): 119-141, 2021; Article no.CJAST.65366
136
Table 5. Mean and standard deviation of physicochemical parameters during bioremediation of crude oil polluted soil
Plot
Treatments
Physicochemical parameters
pH
Temperature
Nitrate
Phosphorus
Potassium
TPH
P1 CTRL 1 - US 6.95±0.20
a
27.62±0.81
a
494.39±24.14
b
2.38±0.26
a
2.11±0.39
a
28.27±28.09
a
P2 CTRL 2 - CS 6.28±0.25
a
28.77±0.96
a
491.06±37.92
ab
2.39±0.31
a
1.99±0.34
a
8612.76±57.80
b
P3 CS+Asp 6.30±0.20
b
28.01±1.03
a
464.44±56.55
ab
2.27±0.39
a
2.61±0.73
a
2418.41±2918.83
ab
P4 CS+Muc 6.25±0.29
a
28.18±1.02
a
453.90±46.57
ab
2.46±0.40
a
2.43±0.64
a
2238.25±2922.68
ab
P5 CS+Asp+Muc 6.43±0.27
a
28.39±1.41
a
472.93±46.52
ab
2.38±0.35
a
3.44±2.07
a
2678.06±3124.12
ab
**means with the same superscript along the columns are not significantly different (p>0.05)
P=- Plot; US = Uncontaminates soil; CS = Contaminated soil; Asp = Aspergillus niger; Muc = Mucor racemosus; SMS = Spent Mushroom Substrate
Table 6. Regression analysis of physiochemical parameters during bioremediation of crude oil polluted soil
Plot
Treatment
pH
Temp
Nitrate
Phosphorus
Potassium
TPH
Regression
equation (Y)
R²
Regression
equation (Y)
R²
Regression
equation (Y)
R²
Regression
equation (Y)
R²
Regression
equation (Y)
R²
Regression
equation (Y)
R²
P1 CTRL 1 - US -0.032x + 7.111 0.195 0.248x + 26.38 0.710 -4.360x + 516.2 0.244 -0.047x + 2.612 0.248 -0.105x + 2.633 0.550 -9.417x + 75.35 0.842
P2 CTRL 2 - CS 0.005x + 6.252 0.003 0.274x + 27.39 0.613 -5.755x + 519.8 0.172 -0.060x + 2.696 0.294 0.006x + 1.961 0.002 -19.49x + 8710 0.853
P3 CS+Asp 0.022x + 6.191 0.093 0.229x + 26.86 0.371 1.034x + 459.2 0.002 -0.065x + 2.615 0.240 0.001x + 2.600 1E-05 -19.49x + 8710 0.737
P4 CS+Muc 0.059x + 5.955 0.324 0.147x + 27.44 0.156 -5.381x + 480.8 0.100 -0.053x + 2.723 0.132 -0.059x + 2.729 0.064 -917.5x + 6826 0.739
P6 CS+Asp+Muc 0.013x + 6.366 0.017 0.342x + 26.68 0.440 -6.041x + 503.1 0.126 -0.042x + 2.595 0.109 -0.171x + 4.294 0.051 -998x + 7668 0.765
P=- Plot; US = Uncontaminated soil; CS = Contaminated soil; Asp = Aspergillus niger; Muc = Mucor racemosus
Table 7. Analysis of bioremediation
Sample ID
Treatments
Initial Conc (mg/kg)Day 1
Final Conc. (mg/kg) Day 56
Amount Remediated (mg/kg)
Actual Amount Remediated (mg/kg)
%Bioremediation (%)
P1 CTRL 1 - US 87.89 2.41 85.48 - -
P2 CTRL 2 - CS 8729.00 8562.46 166.54 81.06 0.32
P3 CS+Asp 8729.00 301.94 8427.06 8341.58 32.98
P4 CS+Muc 8729.00 129.81 8599.19 8513.71 33.66
P6 CS+Asp+Muc 8729.00 286.21 8442.79 8357.31 33.04
Fig. 12. Variation in nitrogen (mg/kg) during bioremediation of crude oil contaminated soil
Fig. 13. Actual % bioremediation assessment during bioremediation of crude oil
Ule et al.; CJAST, 40(1): 119-141, 2021
; Article no.CJAST.65366
137
Fig. 12. Variation in nitrogen (mg/kg) during bioremediation of crude oil contaminated soil
Fig. 13. Actual % bioremediation assessment during bioremediation of crude oil contaminated
soil
; Article no.CJAST.65366
Fig. 12. Variation in nitrogen (mg/kg) during bioremediation of crude oil contaminated soil
contaminated
Ule et al.; CJAST, 40(1): 119-141, 2021; Article no.CJAST.65366
138
4. CONCLUSION AND
RECOMMENDATION
Study showed that bioremediation of crude oil-
contaminated soils with Bioaugmenting fungus
singly may be more effective than combination
with others depending on the type of substrate
used, nature of organism and environmental
conditions prevalent as seen in Mucor
racemosus having higher Bioremediation
potential than when combined with Aspergillus
niger. Notably, Hydrocarbon Utilizing Bacteria
(HUB) and Hydrocarbon Utilizing Fungi (HUF)
which are the key players in Bioremediation have
peak count values on Day 35, this confers that
nutrient renewal on bioremediation sites should
be at interval of 35 days for continuous effective
bioremediation of hydrocarbon pollutants. Also, it
was found that the presence of the crude oil in
the contaminated soil stimulated and sustained
the growth of Hydrocarbon Utilizing Bacteria
(HUB) in the contaminated plots; more so, the
higher growth in the enhanced bio-augmented
plots showed the positive impact Myco (fungal)
bio-augmentation in bioremediation of crude oil
polluted soil. It was further discovered that
treatment plots with higher HUB or HUF had
higher percentage (%) bioremediation; that is,
the higher the sustained HUB and HUF
population, the higher the %Bioremediation.
Summarily, it is therefore recommended that
nutrient renewal on bioremediation site should be
at interval of 35 days for continuous effective
bioremediation of hydrocarbon pollutants. Also,
microbes of high bioremediation potential could
be more effective than consortium of many
hydrocarbon utilizing microbes.
DISCLAIMER
The products used for this research are
commonly and predominantly use products in our
area of research and country. There is absolutely
no conflict of interest between the authors and
producers of the products because we do not
intend to use these products as an avenue for
any litigation but for the advancement of
knowledge. Also, the research was not funded by
the producing company rather it was funded by
personal efforts of the authors.
COMPETING INTERESTS
Authors have declared that no competing
interests exist.
REFERENCES
1. Holliger C, Gaspard S, Glod G, Heijman C,
Schumacher W, Schwarzenbach RP, et al.
Contaminated environments in the
subsurface and bioremediation: Organic
contaminants. FEMS Microbiology
Reviews. 1997;20(3–4):517–523.
2. Nrior RR, Mene GB. Assessment of
bioaugmentation efficiency of Penicillium
Chrysogenum and Aspergillus Nidulans
bioremediation of crude oil spill soil.
Journal of Environmental Science,
Toxicology and Food Technology.
2017;11(8):01-09.
3. Alvarez PJ, Vogel TM. Substrate
interactions of benzene, toluene, and para-
xylene during microbial degradation by
pure cultures and mixed culture aquifer
slurries. Appl. Environ. Microbiol.
1991;57(10):2981–2985.
4. Samanta SK, Singh OV, Jain RK.
Polycyclic aromatic hydrocarbons:
Environmental pollution and
bioremediation. Trends in Biotechnology.
2002;20(6):243–248.
Available:
https://doi.org/10.1016/S0167-
7799(02)01943-1
5. Boffetta P, Jourenkova N, Gustavsson P.
Cancer risk from occupational and
environmental exposure to polycyclic
aromatic hydrocarbons. Cancer Causes &
Control: CCC. 1997;8(3):444–472.
6. Henner P, Schiavon M, Morel JL,
Lichtfouse E. Polycyclic aromatic
hydrocarbon (PAH) occurrence and
remediation methods. Analusis.
1997;25(9–10):56–59.
7. Pilon-Smits E. Phytoremediation. Annu
Rev Plant Biol. 2005;56:15–39.
8. Liang B, Lehmann J, Solomon D, Kinyangi
J, Grossman J, O’neill B, et al. Black
carbon increases cation exchange capacity
in soils. Soil Science Society of America
Journal. 2006;70(5):1719–1730.
9. Bojes HK, Pope PG. Characterization of
EPA’s 16 priority pollutant polycyclic
aromatic hydrocarbons (PAHs) in tank
bottom solids and associated
contaminated soils at oil exploration and
production sites in Texas. Regulatory
Toxicology and Pharmacology.
2007;47(3):288–295.
10. Gao Y, Collins CD. Uptake pathways of
polycyclic aromatic hydrocarbons in white
Ule et al.; CJAST, 40(1): 119-141, 2021; Article no.CJAST.65366
139
clover. Environmental Science &
Technology. 2009;43(16):6190–6195.
11. Cook RL, Landmeyer JE, Atkinson B,
Messier JP, Nichols EG. Field note:
Successful establishment of a
phytoremediation system at a petroleum
hydrocarbon contaminated shallow aquifer:
trends, trials, and tribulations. International
Journal of Phytoremediation.
2010;12(7):716–732.
12. Boonsaner M, Borrirukwisitsak S,
Boonsaner A. Phytoremediation of BTEX
contaminated soil by Canna$\times$
generalis. Ecotoxicology and
Environmental Safety. 2011;74(6):1700–
1707.
13. Hreniuc M, Coman M, Cioruţa B.
Considerations regarding the soil pollution
with oil products in Săcel-Maramureş.
Scientific Research & Education in the Air
Force-AFASES. 2015;2.
14. Zahed MA, Aziz HA, Isa MH, Mohajeri L.
Enhancement biodegradation of n-alkanes
from crude oil contaminated seawater.
International Journal of Environmental
Research. 2010;4(4):655–664.
15. Masakorala K, Yao J, Chandankere R, Liu
H, Liu W, Cai M, et al. A combined
approach of physicochemical and
biological methods for the characterization
of petroleum hydrocarbon-contaminated
soil. Environmental Science and Pollution
Research. 2014;21(1):454–463.
16. Tang JC, Wang RG, Niu XW, Wang M,
Chu HR, Zhou QX. Characterisation of the
rhizoremediation of petroleum-
contaminated soil: Effect of different
influencing factors. Biogeosciences.
2010;7(12):3961–3969.
17. Kang JW. Removing environmental
organic pollutants with bioremediation and
phytoremediation. Biotechnology Letters.
2014;36(6):1129–1139.
18. Nichols EG, Cook RL, Landmeyer JE,
Atkinson B, Malone DR, Shaw G, et al.
Phytoremediation of a Petroleum-
Hydrocarbon Contaminated Shallow
Aquifer in Elizabeth City, North Carolina,
USA. Remediation Journal.
2014;24(2):29–46.
19. Azubuike CC, Chikere CB, Okpokwasili
GC. Bioremediation techniques–
classification based on site of application:
Principles, advantages, limitations and
prospects. World Journal of Microbiology
and Biotechnology. 2016;32(11):180.
20. Ogbonna DN. Application of biological
methods in the remediation of oil polluted
environment in Nigeria. Journal of
Advances in Biology & Biotechnology.
2018;17(4):1-10.
21. Frutos FJG, Escolano O, García S, Babín
M, Fernández MD. Bioventing remediation
and ecotoxicity evaluation of
phenanthrene-contaminated soil. Journal
of Hazardous Materials. 2010;183(1–
3):806–813.
22. Amro MM. Treatment techniques of oil-
contaminated soil and water aquifers.
International Conf. on Water Resources &
Arid Environment. 2004;1–11.
23. Bidoia ED, Montagnolli RN, Lopes PRM.
Microbial biodegradation potential of
hydrocarbons evaluated by colorimetric
technique: A case study. Appl Microbiol
Biotechnol. 2010;7:1277–1288.
24. Djelal H, Amrane A. Biodegradation by
bioaugmentation of dairy wastewater by
fungal consortium on a bioreactor lab-scale
and on a pilot-scale; 2013.
25. Okerentugba PO, Orji FA, Ibiene AA,
Elemo GN. Spent mushroom compost for
bioremediation of petroleum hydrocarbon
polluted soil: A review. Global Advanced
Research Journal of Environmental
Science and Toxicology. 2015;4(1):001–
007.
26. Mohammadi-Sichani MM, Assadi MM,
Farazmand A, Kianirad M, Ahadi AM,
Ghahderijani HH. Bioremediation of soil
contaminated crude oil by Agaricomycetes.
Journal of Environmental Health Science
and Engineering. 2017;15(1):8.
27. Ogbonna DN, Ngah SA, Okparanma RN,
Ule O, Nrior RR. Percentage
Bioremediation Assessment of Spent
Mushroom Substrate (SMS) and Mucor
racemosus in Hydrocarbon Contaminated
Soil. Journal of Advances in Microbiology.
2021;20(12):1-21.
28. Bockelmann A, Zamfirescu D, Ptak T,
Grathwohl P, Teutsch G. Quantification of
mass fluxes and natural attenuation rates
at an industrial site with a limited
monitoring network: A case study. Journal
of Contaminant Hydrology. 2003;60(1–
2):97–121.
29. Wegwu MO, Uwakwe AA, Anabi MA.
Efficacy of enhanced natural attenuation
(land farming) technique in the remediation
of crude oil-polluted agricultural land. Arch
Appl Sci Res. 2010;2(2):431–442.
Ule et al.; CJAST, 40(1): 119-141, 2021; Article no.CJAST.65366
140
30. Lee EH, Kang YS, Cho KS. Bioremediation
of diesel-contaminated soils by natural
attenuation, biostimulation and
bioaugmentation employing Rhodococcus
sp. EH831. Korean J Microbiol Biotechnol.
2011;39(1):86–92.
31. Ogbonna DN, Nrior RR, Ezinwo FE.
Bioremediation Efficiency of Bacillus
amyloliquefaciens and Pseudomonas
aeruginosa with the Nutrient Amendment
on Crude Oil Polluted the Soil.
Microbiology Research Journal
International. 2019;1–13.
Available
:
https://doi.org/10.9734/mrji/2019/
v29i530175
32. Toogood TA. Effect of oil spill on the
physical properties of soils. In: J.A.
Toogood (ed). The reclamation of
agricultural soil after oil spills. Part 1
Research, Alberta Institute of Pedology,
Canada, Publication No. 1977;11-77:108-
121.
33. Menkit MC, Amechi AK. Evaluation of first
and second order degradation rates and
biological half-lives in crude oil polluted
soil. Asian Journal of Biotechnology and
Genetic Engineering. 2019;2(1):1-11.
34. Nrior RR, Odokuma LO. Ultimate
biodegradability potential of
trichloroethylene (TCE) used as degreaser
in marine, brackish and fresh water.
Journal of Environmental Sciences,
Toxicology and Food Technology.
2015;9:80-89. 18.
35. Nrior RR, Echezolom C. Assessment of
percentage bioremediation of Petroleum
Hydrocarbon polluted soil with
biostimulating agents. Journal of Current
Studies in Comparative Education,
Science and Technology. 2017;3(1):203-
215.
36. APHA. Standard methods for the
examination of water and waste water.
20th ed. APHA-AWWA-WPCF.
Washington DC; 1998.
37. Chikere CB, Okpokwasili GC, Chikere BO.
Bacterial diversity in a tropical crude oil
polluted soil undergoing bioremediation.
African Journal of Biotechnology. 2009;
8(11):2535-2540. 20.
38. Ollivier B, Magot M. Petroleum
Microbiology. Washington, DC: ASM:
12/08/2017; Heat of Combustion of Fuels.
2005;1.
39. Kidd S, Halliday C, Alexiou H, Ellis D.
Description of medical fungi (3rd ed).
Adelaide, Australia. 2016;232-235.
40. Cheesbrough M. District Laboratory
Practice in Tropical Countries. 2006;2-5.
41. Harms H. Bioavailability and
Bioaccessibility as Key Factors in
Bioremediation. In M. Moo-Young (Ed.),
Comprehensive Biotechnology (Second
Edition). 2011;83–94. Academic Press.
Available:https://doi.org/10.1016/B978-0-
08-088504-9.00367-6
42. Liu GM, Yang JS, Yao RJ. Electrical
conductivity in soil extracts: Chemical
factors and their intensity1 1project
supported by the national basic research
program of China (No. 2005CB121108),
the national natural science foundation of
China (No. 40371058), and the national
high technology research and
development program of China (863
Program) (No. 2002AA2Z4061).
Pedosphere. 2006;16(1):100–107.
Available:https://doi.org/10.1016/S1002-
0160(06)60031-3
43. Dalefield R. Chapter 18—Industrial and
occupational toxicants. In R. Dalefield
(Ed.), Veterinary Toxicology for Australia
and New Zealand. 2017;333–341).
Elsevier.
Available:https://doi.org/10.1016/B978-0-
12-420227-6.00017-7
44. Ayuba SA, John C, Obasi MO. Effects of
organic manure on soil chemical properties
and yield of ginger—Research note.
Nigerian Journal of Soil Science.
2005;15:136–138.
https://doi.org/10.4314/njss.v15i1.37461
45. Oa E, Fi A. Bioremediation of petroleum
hydrocarbons from crude oil-contaminated
soil with the earthworm: Hyperiodrilus
Africanus. 3 Biotech. 2015;5(6):957–965.
Available:https://doi.org/10.1007/s13205-
015-0298-1
46. Benyahia F, Embaby AS. Bioremediation
of crude oil contaminated desert soil: effect
of biostimulation, bioaugmentation and
bioavailability in biopile treatment systems.
International Journal of Environmental
Research and Public Health. 2016;13(2).
Available:https://doi.org/10.3390/ijerph130
20219
47. Ebuehi OaT, Abibo IB, Shekwolo PD,
Sigismund KI, Adoki A, Okoro IC.
Remediation of crude oil contaminated soil
by enhanced natural attenuation
technique. Journal of Applied Sciences
and Environmental Management.
2005;9(1):Article 1.
Ule et al.; CJAST, 40(1): 119-141, 2021; Article no.CJAST.65366
141
Available:https://doi.org/10.4314/jasem.v9i
1.17265
48. Wang Y, Tam NFY. Chapter 16—Microbial
remediation of organic pollutants. in C.
Sheppard (Ed.), World Seas: An
Environmental Evaluation (Second
Edition). 2019;283–303).
Academic Press.
Available:https://doi.org/10.1016/B978-0-
12-805052-1.00016-4.
_________________________________________________________________________________
© 2021 Ule et al.; This is an Open Access article distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium,
provided the original work is properly cited.
Peer-review history:
The peer review history for this paper can be accessed here:
http://www.sdiarticle4.com/review-history/65366
