Petroleum & Environmental

Biotechnology

Agarry et al., J Pet Environ Biotechnol 2012, 3:3

http://dx.doi.org/10.4172/2157-7463.1000120

Research Article

Open Access

Enhanced Bioremediation of Soil Artificially Contaminated with Kerosene:

Optimization of Biostimulation Agents through Statistical Experimental
Design
Agarry SE1*, Owabor CN2 and Yusuf RO3
1
2
3

Biochemical Engineering and Biotechnology Laboratory, Department of Chemical Engineering, Ladoke Akintola University of Technology, Ogbomoso, Nigeria
Department of Chemical Engineering, University of Benin, Benin-City, Nigeria
Environmental Engineering Laboratory, Department of Chemical Engineering, University of Technology, Johor Bahru, Malaysia

Abstract
The main goals of this work were to study the enhanced bioremediation of soil artificially contaminated with
kerosene via biostimulation strategy, evaluate the influence of biostimulating agents on the rate of degradation
and to optimize the biostimulating agents for maximum kerosene removal. The study was carried out by artificially
contaminating an un-impacted tropical soil with 10% (w/w) kerosene oil in earthen pots and various concentrations
of NPK fertilizer, Tween 80 and hydrogen peroxide were added and then incubated for six weeks remediation period.
To optimize the range of experimentation, Response Surface Methodology (RSM) with Box Behnken Design (BBD)
was used with three factors and three levels of NPK fertilizer, Tween 80 and hydrogen peroxide as independent
variables and kerosene oil (total petroleum hydrocarbon) removal as dependent variable (response). The results
showed that there were significant variations in the kerosene oil biodegradation pattern with respect to NPK fertilizer,
Tween 80 and hydrogen peroxide. A statistically significant (P < 0.0001) second-order quadratic regression model
for kerosene oil removal (using Design-Expert Statistical program (v. 6.0.8) with a coefficient of determination,
R (= 0.9992) was obtained. Numerical optimization technique based on desirability function was carried out to
optimize the bioremediation process. The optimum values for biostimulating agents to achieve a predicted maximum
kerosene removal of 75.06% were found to be: NPK fertilizer, 4.30 g (equivalent to 0.0215 g/kg); Tween 80, 10.03
mg/l and hydrogen peroxide, 1.13 g/l. At this optimum point, the observed kerosene oil removal was found to be
73.95%. Thus, biostimulation of indigenous microbial density and activity can reduce the period for remediation of
contaminated environment and subsequently the cost of remediation. Response Surface Methodology (RSM) is a
reliable and powerful tool for modeling and optimizing of kerosene oil bioremediation processes.

Keywords: Bioremediation; Biostimulation; Kerosene; NPK

fertilizer; Tween 80; Hydrogen peroxide; Response surface methodology

Introduction
Petroleum products obtained from the refining of crude oil are
some of the most widely used chemicals in society today. Kerosene is a
colourless flammable hydrocarbon liquid obtained from the fractional
distillation of petroleum at 150C and 275C [1,2]. Kerosene contains
paraffins (alkanes), cycloparaffins (cycloalkanes), aromatics and
olefins from approximately C9 to C20 [3]. It is used to run many types
of engines, lamps, heaters and stoves. The invention of the internal
combustion engine and its fast adoption in all transport forms enlarged
the employment of this natural resource, thus increasing its demand
production, transport, stockpiling, and distribution, as well as the raw
oil and its by-products. All these activities involve pollution risks that
can be minimized, but not totally eliminated, causing several problems
for the environment [4].
Kerosene has many toxic effects on plants, animals and humans [1]
and possesses moderate to high acute toxicity to biota with productspecific toxicity related to the type and concentration of aromatic
compounds [5]. Generally, petroleum contamination results from
leakages of underground and above ground storage tanks, spillage
during transport of petroleum products, tanker accidents, unplanned
releases and current industrial processes [6]. The application of
biotechnological processes involving microorganisms, with the
objective of solving environmental pollution problems, is rapidly
growing, in recent decades, where petroleum and its by-products
are concerned. Bioremediation processes, which take advantage of
J Pet Environ Biotechnol
ISSN: 2157-7463 JPEB, an open access journal

microbial degradation of organic and inorganic substances, can be

defined as the use of biological systems to catalyze the destruction or
transformation of various chemicals to less harmful forms [7].
It is known that biodegradation efficiencies of petroleum
hydrocarbons in soil can be limited by physicochemical as well as
biological factors, such as nutrients, pH, temperature, oxygen, number
and type or species of microorganisms [8-10]. Also, oil spills result
in an imbalance in the carbonnitrogen ratio at the spill site, because
crude-oil is essentially a mixture of carbon and hydrogen. This causes
a nitrogen deficiency in an oil-soaked soil, which retards the growth
of bacteria and the utilization of carbon source(s). In addition to a
nitrogen deficiency in oil-soaked soil, certain nutrients like phosphorus
may be growth-rate limiting [11]. Furthermore, large concentrations
of biodegradable organics in the top layer of agricultural soils deplete
oxygen reserves in the soil and slow down the rates of oxygen diffusion
to deeper layers [12].

*Corresponding author: Agarry SE, Biochemical Engineering and Biotechnology

Laboratory, Department of Chemical Engineering, Ladoke Akintola University of
Technology, Ogbomoso, Nigeria, E-mail: sam_agarry@yahoo.com
ReceivedFebruary 25, 2012; Accepted April 21, 2012; Published April 23, 2012
Citation: Agarry SE, Owabor CN, Yusuf RO (2012) Enhanced Bioremediation
of Soil Artificially Contaminated with Kerosene: Optimization of Biostimulation
Agents through Statistical Experimental Design. J Pet Environ Biotechnol 3:120.
doi:10.4172/2157-7463.1000120
Copyright: 2012 Agarry SE, et al. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.

Volume 3 Issue 3 1000120

Citation: Agarry SE, Owabor CN, Yusuf RO (2012) Enhanced Bioremediation of Soil Artificially Contaminated with Kerosene: Optimization of
Biostimulation Agents through Statistical Experimental Design. J Pet Environ Biotechnol 3:120. doi:10.4172/2157-7463.1000120

Page 2 of 8
Petroleum hydrocarbon pollution tends to persist in soils until
remediation measures, involving the application of nutrients, are
resorted to, because oxygen and nitrogen are limiting factors in all
types of petroleum degradation. Oxygen levels must be high enough
for the breakdown of hydrocarbons. Thus, pollutant degradation rates
can be enhanced by the addition of nutrients, oxygen, and primary
substrates into the contaminated systems. This could increase the
populations of indigenous microorganisms and thus improve the
efficiency of pollutant biodegradation. In biostimulation technology,
nutrient supplementation for petroleum hydrocarbon degradation has
traditionally focused on addition of nitrogen (N) and phosphorus (P),
either organically or inorganically. The effects of nutrients (i.e. NPK),
aeration and biostimulation of indigenous soil microorganisms and
inoculation of extraneous microbial consortia on the bioremediation
of oil contaminated soil have been investigated [13-15]. Interest has
recently been shown in the use of oxygen-release compounds (ORCs)
to promote the direct oxidation of pollutants and, at the same time,
to increase aerobic microbial degradation [16,17]. As most of the
bioremediation methods are aerobic processes, due to its greater
efficiency, both the oxygen generated and delivery of oxygen to soil (in
situ bioremediation technologies) is crucial to success.
The introduction of pure oxygen rather than air can significantly
increase the dissolved oxygen (DO) concentrations [18]. Injection of
hydrogen peroxide (H2O2) is one of the methods to increase the DO
concentrations. A significant portion of contaminants in soils has
been found to be oxidized by H2O2 without any addition of soluble
iron [18-20] and the mineral catalyzed Fenton-like reaction was
proposed to describe the oxidation occurring in the natural soils. One
of the advantages of applying Fenton-like oxidation for contaminant
oxidation is that the produced oxygen during the decomposition of H2O2
would increase the DO concentration. This would enhance the aerobic
biodegradation rate of contaminant [21]. Application of surfactants
to soil contaminated with petroleum hydrocarbons can potentially
reduce the interfacial tension, increase its solubility and bioavailability,
and thus facilitate their biodegradation [22,23]. Recently, statistical
design of experiment technique has been successfully applied in many
fields [4,24-26] to provide information about direct effects, mutual
interaction effects and curvilinear variable effects.
Biostimulation can be considered as an appropriate remediation
technique for kerosene removal in soil and requires the evaluation
of both the intrinsic degradation capacities of the autochthonous
microflora and the environmental parameters involved in the kinetics
of the in situ process. One of those parameters is aeration, which
can be improved in bioremediation systems by the use of Oxygen
Release Compounds (ORC). Biostimulation in systems controlling
different physical and chemical factors has been well documented
[27-29]. The biological removal of petroleum commercial products,
such as kerosene has been reported [2,30-32] however, there is a lack
of information on the use of ORC for stimulation of autochthonous
microflora of kerosene contaminated soils. More also, available
information regarding the effects of biodegradable surfactants
addition on enhanced biodegradation or removal of kerosene oil as
well as information on the optimization of these biostimulating factors
required for the enhanced biodegradation is very limited.
Therefore, the objective of this work was to study the bioremediation
of soil artificially contaminated with kerosene via biostimulation
strategy as a function of nutrients (NPK fertilizer), oxygen release
J Pet Environ Biotechnol
ISSN: 2157-7463 JPEB, an open access journal

compound (hydrogen peroxide) and surfactant (Tween 80) through

Response Surface Methodology (RSM) using full-factorial Box Behnken
design in order to evaluate the influence of these biostimulating factors
on the rate of degradation and to optimize the factors for maximum
kerosene removal.

Materials and Methods

Soil sample and characterization
An un-impacted soil samples from Ladoke Akintola University of
Technology Agricultural Farms, Ogbomoso, were collected from the
surface layer of the vadose zone 15-30 cm below land surface. The soil
samples were air dried, homogenized, passed through a 2 - mm (pore
size) sieve and stored in a polythene bag at room temperature. Soil
samples were characterized for their physicochemical and microbial
parameters according to standard methods. Total organic carbon
and Total Nitrogen of soil were determined using Walkley-Black and
Macro-Kjeldahl methods respectively [33,34]. Soil pH was determined
using pH meter fitted with a combined glass pH and reference electrode.
Soil moisture content was determined by evaporation on Whatman
filter paper NO 1 (BDH Chemicals England) at 103 to 105C in an
electrical oven. Available phosphorus was determined using Bray NO 1
Method [33,34]. The Total Hydrocarbon Degrading Bacteria (THDB)
populations were determined by the vapour phase transfer method
[35].

Chemicals
The kerosene oil was purchased from a local petroleum station in
Ogbomoso, Nigeria. Hydrogen peroxide (H2O2) 50% (w/v), (a product
of Merck, USA) and hexane solvent (BDH Chemicals, England) used for
extraction of oil from soil were bought from a chemical store in Lagos,
Nigeria and used as received. This study employed a biodegradable
non ionic surfactant Tween 80 manufactured by Sigma-Aldrich, USA,
which has an average molecular weight of 1310 and a Critical Micelle
Concentration (CMC) value of 15 mg/l. NPK fertilizer (20:10:10) was
purchased from an agro-chemical store in Ogbomoso, Nigeria.

Microcosms preparation and bioremediation experimentation

To optimize the range of experimentation for 23 full-factorial BoxBehnken design, the following experiments were performed in earthen
pots maintained at room temperature. Soil samples (200 g) were placed
in earthen pots (microcosm) and were artificially contaminated with
kerosene fuel (obtained from a local commercial source) to a level of
10 % w/w. The kerosene-contaminated soil in each earthen pot was
amended with different amounts of NPK fertilizer (2 - 10 g, equivalent
to 0.01 0.05 g/kg), Tween 80 (5 - 25 mg/l) and hydrogen peroxide
(0.5 - 2.5 g/l), respectively. Soil used as control was not amended with
any biostimulating agents. In total, 16 microcosms were settled and
incubated for 42 days. All reactors were mixed manually once per week
to enhance oxygenation, and kept moist during the 42 day experimental
period. Samples were withdrawn at intervals of one week for residual
kerosene or total petroleum hydrocarbon analysis.

Experimental design and data analysis

The Box-Behnken factorial experimental design employed had
three independent variables viz, NPK (20:10:10) fertilizer, Tween 80
(surfactant) and hydrogen peroxide (ORC). Each of the independent
variables was studied at three levels (1, 0, +1), with 17 experimental
runs and one control. The levels were selected based on preliminary

Volume 3 Issue 3 1000120

Citation: Agarry SE, Owabor CN, Yusuf RO (2012) Enhanced Bioremediation of Soil Artificially Contaminated with Kerosene: Optimization of
Biostimulation Agents through Statistical Experimental Design. J Pet Environ Biotechnol 3:120. doi:10.4172/2157-7463.1000120

Page 3 of 8
study results and literature. The variables optimized were NPK fertilizer
(from 2 - 6 g or 0.01 0.03 g/kg), Tween 80 (5 15 mg/l) and hydrogen
peroxide (0.5 1.5 g/l), respectively Table 1. Change in kerosene
oil removal was considered as experimental response. Efficiency of
kerosene removal was assessed after 42 days. Table 3 shows the coded
and actual values of factors and levels used in the experimental design.
Kerosene contaminated soil without biostimulation was also assayed as
a control. The statistical software Design Expert 6.0.8, (Stat-Ease Inc.,
Minneapolis, USA) was used to evaluate the analysis of variance (P <
0.05) to determine the significance of each term in the fitted equations
and to estimate the goodness of fit in each case.

in the form of NPK inorganic fertilizer (20:10:10) to provide the proper

nutrients required for the bioremediation process.

Natural bioattenuation and enhanced bioremediation

After performing 17 runs of the Box-Behnken Design (BBD) and
one control, the results of the statistical experiment were analyzed with
regard to the coded design matrix Table 2. The regression equation
shows that the kerosene oil degradation rate was an experimental
function of test variables in coded units. The comparison of kerosene
oil enhanced bioremediation and natural bioattenuation for each run
is shown in Table 3 as well as in Figure 1. Table 3 and Figure 1 show
that on the 42 day, kerosene oil content had decreased in all earthen pot
reactors. In control, natural biodegradation (natural bioattenuation)
removed 38.5 per cent of petroleum hydrocarbons. The reduction in
hydrocarbon content of earthen pot reactors containing biostimulants
was much higher (Table 3 and Figure 1) in the same period. These
results indicate that the addition of biostimulants increased the rate
of biodegradation. A considerable decrease in oil reduction was
observed in run 12, with the highest amount of surfactant (Tween 80)
and oxygen releasing compound (hydrogen peroxide), the residual oil
reached 22.11 per cent of the initial kerosene oil concentration. Yuting
et al. [37] showed that natural attenuation removed 13 per cent of
crude oil after 33 day incubation. When the soil was supplemented with
nutrients (nitrogen and phosphorus) and biomass, 26.3 per cent of the
crude oil was removed.

Estimation of total petroleum hydrocarbon (residual kerosene oil)

Total petroleum hydrocarbon (TPH) was extracted from 10 g of soil
with 50 ml of hexane [36]. The extract was dried at room temperature
by evaporation of the hexane solvent under a gentle nitrogen stream in
a fume hood. After evaporation of the solvent, the amount of residual
TPH was determined gravimetrically [36] by reading absorbance at 400
nm using visible range spectrophotometer (Model 6100 PYE UNICAM
instruments England) and estimating the concentration from the
standards curve, obtained from hexane extracts of fresh kerosene oil at
different concentrations.

Results and Discussion

One of the major factors limiting degradation of hydrocarbons

is their low availability to the microbial cells [38,39]. In addition,
hydrocarbon-oxidizing potential has also been shown to increase
with hydrocarbon exposure [40]. Thus, as seen in Figure 1 (at 10 g per
kg soil kerosene oil concentrations), run numbers 1 and 3 (at lower
concentration of NPK and H2O2), and run numbers 11 and 12 (at higher
concentration of NPK and H2O2) had same remediation conditions
but with different surfactant (Tween 80) concentration, results shows
that addition of surfactant can enhance kerosene degradation. Similar

Soil parameters
The determined soil parameters values are as follows: moisture
content - 5.95 0.05 (%), total nitrogen - 0.25 0.04 (%), available
phosphorus - 0.12 0.02 (%), potassium - 0.31 0.05 (%), total organic
carbon - 1.21 0.03 (%), pH 5.9 0.2, and Total hydrocarbon-degrading
bacteria (THDB) - 3.7 105cfu-g-1. The soil characterization showed
that the soil did not fulfil the nutrient (NPK) requirements for an
efficient biodegradation process. Therefore, these elements were added
Dependent variable Low Level (

1) Medium Level(0) High Level

(+ 1)

Dependent variable Low Level (

1) Medium Level(0) High Level

(+ 1)

NPK Fertilizer (A)

2.0

4.0

6.0

Tween 80 (B )

5.0

10

15

Hydrogen Peroxide ( C)

0.5

1.0

1.5

Table 1: Experimental range and the levels of the variables.

Run number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18 (control)

NPK Fertilizer (A)

Tween 80 (B)

Hydrogen Peroxide (C)

Code

Value(g)

Code

Value(g/l)

Code

Value(g)

-1
+1
-1
+1
-1
+1
-1
+1
0
0
0
0
0
0
0
0
0
-

2
6
2
6
2
6
2
6
4
4
4
4
4
4
4
4
4
-

-1
-1
+1
+1
0
0
0
0
-1
+1
-1
+1
0
0
0
0
0
-

5
5
15
15
10
10
10
10
5
15
5
15
10
10
10
10
10
-

0
0
0
0
-1
-1
+1
+1
-1
-1
+1
+1
0
0
0
0
0
-

1.0
1.0
1.0
1.0
0.5
0.5
1.5
1.5
0.5
0.5
1.5
1.5
1.0
1.0
1.0
1.0
1.0
-

Table 2: Coded and uncoded full-factorial Box-Behnken design for the three independent variables.

J Pet Environ Biotechnol

ISSN: 2157-7463 JPEB, an open access journal

Volume 3 Issue 3 1000120

Citation: Agarry SE, Owabor CN, Yusuf RO (2012) Enhanced Bioremediation of Soil Artificially Contaminated with Kerosene: Optimization of
Biostimulation Agents through Statistical Experimental Design. J Pet Environ Biotechnol 3:120. doi:10.4172/2157-7463.1000120

Page 4 of 8
Percent Kerosene Removal
Experimental Run

Observed Value
Predicted Value

Observed Value Predicted

Value

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18

65.15
67.43
65.06
68.43
68.02
68.96
67.04
72.06
69.54
69.88
69.92
71.75
74.73
74.85
75.00
74.66
74.92
38.50

64.95
67.31
65.18
68.63
68.04
68.90
67.09
72.04
69.71
69.74
70.06
71.58
74.83
74.83
74.83
74.83
74.83
-

Table 3: Experimental design and results for kerosene removal from

contaminated soil.

Kerosene Oil Removal (%)

80
70
60
50

accelerate the initial oil degradation rate and may shorten the period to
clean up contaminated environments.
The accelerating effect of amendment is stronger when nutrient
availability is a limiting factor in the biodegradation of oil. Similar
observations have been reported for the use of nitrogen and
phosphorus in the bioremediation of environment contaminated with
petroleum hydrocarbons [2,38,46,48]. Bioremediation efficiency is
a function of the microbial viability in the natural environment [49].
Microorganisms need nutrient to grow. Hence, biodegradation of
hydrocarbons in the natural environment is limited by poor growth
rate of microorganisms caused by nutrient deficiencies, especially in
nitrogen and phosphorus [40,50]. Therefore, when bioremediation is
conducted suitable nitrogen and phosphorus are usually applied to the
contaminated environment to stimulate biodegradation [51].

Second order polynomial regression model and statistical

analysis
The experimental data were fitted to a second order polynomial
regression model (Equation 1) containing 3 linear, 3 quadratic and 3
interaction terms [52] using the same experimental design software
to derive the equation (Equation (1)) for kerosene oil removal from
contaminated soil.
Y= 0 + 1 A + 2 B + 3 C + 11 A2 + 22 B2 + 33 C2 + 12 AB + 13 AC
+ 23 BC
(1)

40
30
20
10
0
1

Natural Bioattenuation

10

11

12

13

14

15

16

17

Experimental Run
Enhanced Bioremediation

Figure 1: Comparison of percent kerosene oil removal due to natural

bioattenuation and enhanced bioremediation. 1 = NPK (-1) + Tween 80 (-1) +
H2O2 (0), 2 = NPK (+1) + Tween 80 (-1) + H2O2 (0), 3 = NPK (-1) + Tween 80
(+1)+ H2O2 (0), 4 = NPK (-1) + Tween 80 (+1) + H2O2 (0),5 = NPK (-1) + Tween 80
(0) + H2O2 (-1), 6 = NPK (+1) + Tween 80 (0) + H2O2 (-1), 7 = NPK (-1) + Tween
80 (0) + H2O2 (+1), 8 = NPK (+1) + Tween 80 (0) + H2O2 (+1), 9 = NPK (0) +
Tween 80 (-1) + H2O2 (-1), 10 = NPK (0) + Tween 80 (+1) + H2O2 (-1), 11 = NPK
(0) + Tween 80 (-1) + H2O2 (+1), 12 = NPK (0) + Tween 80 (+1) + H2O2 (+1), 13 =
NPK (0) + Tween 80 (0) + H2O2 (0), 14 = NPK (0) + Tween 80 (0) + H2O2 (0), 15
= NPK (0) + Tween 80 (0) + H2O2 (0), 16 = NPK (0) + Tween 80 (0) + H2O2 (0),
17= NPK (0) + Tween 80 (0) + H2O2 (0).

observations have been reported for the use of non-ionic surfactant

for the remediation of environment contaminated with petroleum
hydrocarbons [18,41,42].
Effect of different concentrations of hydrogen peroxide
supplementation were investigated at the same condition of NPK
and Tween 80 (run numbers 5 and 7, and run numbers 10 and 12)
and the findings demonstrated that addition of hydrogen peroxide
(ORC) can enhance the bioremediation process of soil contaminated
with kerosene. This is in agreement with earlier workers observations
[18,43,44]. Furthermore, at the Traverse city, Michigan, bioremediation
of a contaminated site was achieved by an increase in the oxygen
concentration in water through addition of hydrogen peroxide and
was observed to positively affect the rate of biodegradation [45]. On
the other hand; run numbers 1 and 2 and run numbers 5 and 6 through
similar condition but with different amount of NPK were tested and the
results shows that extra amount of NPK (from 2 to 4 g/l. equivalent to
0.01 0.02 g/kg) can improve kerosene removal from contaminated
soil. The results suggest that high dose nutrient amendment can

J Pet Environ Biotechnol

ISSN: 2157-7463 JPEB, an open access journal

where 0 is the value of the fixed response at the centre point of

the design; 1, 2, 3 are linear coefficients; 12, 13, 23 are quadratic
coefficients; are the interaction effect coefficients regression terms,
respectively; A, B and C are the levels of independent variables The
significance of each coefficient in the equation was determined by
F-test and P-values. F-test indicated that all the factors and interactions
considered in the experimental design are statistically significant (P
< 0.05) at the 95 per cent confidence level. The regression equation
obtained after analysis of variance gives the level of kerosene removal
as a function of the different biostimulation variables: NPK, Tween 80,
and Hydrogen peroxide. All terms regardless of their significance are
included in the following Equation (2):
Y = 74.83 +1.45A + 0.38B + 0.55C 4.78A2 3.53B2 1.03C2 +
0.27AB + 1.02AC + 0.37BC
(2)
where A is NPK concentration, B is Tween concentration; C is
Hydrogen peroxide concentrations. To test the fit of the model, the
regression equation and determination coefficient (R2) were evaluated
(Table 4). The model F-value of 515.92 implies the model is significant.
There is only a 0.01 per cent chance that a model F-value, this large
could occur due to noise alone. The low probability value (<0.0001)
indicates that the model is significant. The value of the determination
coefficient (R2 = 0.9985) being a measure of goodness of fit to the
model indicates a high degree of correlation between the observed
value and predicted values. The determination coefficient (R= 0.9992),
suggests that more than 99.92% of the variance is attributable to the
variables and indicated a high significance of the model. Thus, 0.08% of
the total variance cannot be explained by the model. The fitted model is
considered adequate if the F-test is significant (P < 0.05). The Analysis
Of Variances (ANOVA) quadratic regression model demonstrated
that the model was highly significant, as was evident from the very low
probability (P < 0.0001) of the F - test and insignificant result from
the Lack of Fit model (P = 0.1186). The Lack of Fit test is performed

Volume 3 Issue 3 1000120

Citation: Agarry SE, Owabor CN, Yusuf RO (2012) Enhanced Bioremediation of Soil Artificially Contaminated with Kerosene: Optimization of
Biostimulation Agents through Statistical Experimental Design. J Pet Environ Biotechnol 3:120. doi:10.4172/2157-7463.1000120

Page 5 of 8
by comparing the variability of the current model residuals to the
variability between observations at replicate settings of the factors. The
Lack of Fit F -value of 3.72 implies the Lack of Fit is not significant
relative to the pure error. There is an 11.86 per cent chance that a Lack
of Fit F -value this large could occur due to noise.
The Lack of Fit is designed to determine whether the selected
model is adequate to describe the observed data, or whether a more
complicated model should be used. The Predicted R-Squared value of
0.9817 is in reasonable agreement with the Adjusted R-Squared value of
0.9966. Adequate Precision measures the signal to noise ratio. A ratio >
4 is desirable. The ratio of 63.404 obtained in this research indicates an
adequate signal. This model can be used to navigate the design space.
The coefficient of variation (CV) as the ratio of the standard error of
estimate to the mean value of the observed response is a measure of
reproducibility of the model, generally a model can be considered
reasonably reproducible if its CV is not greater than 10 per cent. Hence,
the low variation Coefficient value (CV = 0.29 per cent) obtained
indicates a high precision and reliability of the experiments. The
coefficient of the model (parameter estimation) and the corresponding
P-values are presented in Table 5. The significance of regression
coefficients was considered, ignoring those with an insignificant effect
on the response at a significance level of 95%. The P-values of the
regression coefficients suggest that among the test variables, linear,
quadratic and interaction effects of NPK fertilizer, Tween 80 and
hydrogen peroxide are highly significant. The insignificant effects
(factors and interactions) with P-values higher than 0.05, were ignored.
In this study, A, B, C, A2, B2, C2, AB, AC and BC are significant model
terms.
Thus, statistical analysis of all the experimental data showed that
NPK fertilizer, Tween 80 and hydrogen peroxide concentration had
a significant effect on oil removal during the study. Moreover, it is
observed that NPK fertilizer (nutrients) exerted more pronounced
linear effect (higher coefficient values) on kerosene removal. That
is, kerosene removal was mostly and positively influenced by NPK
fertilizer (nutrients) followed by hydrogen peroxide (ORC) and
Tween 80 (surfactant). The strong influence of nutrients on petroleum
Source
Model
Residual (error)
Lack of Fit
Pure Error
Correlation Total

Sum of squares

DF Mean square P-value

191.61
0.64
0.21
0.076
191.81

9
7
3
4
17

21.29
0.091
0.071
0.019

F-value

515.92 <0.0001
3.72

0.1186

R2= 0.9985 Adj R2 = 0.9966 Predicted R2= 0.9817 Adeq. Precision = 63.404
Table 4: Analysis of variance (ANOVA) for the quadratic response surface model
fitting to the biodegradation data of kerosene oil.
Variables Coefficient Standard
value
error
0
1
2
3
11
22
33
12
13
23

74.83
1.45
0.38
0.55
-4.78
-3.53
-1.03
0.27
1.02
0.37

0.091
0.072
0.072
0.072
0.099
0.099
0.099
0.100
0.100
0.100

F-value

P-value

Remarks

515.92
408.31
28.74
57.85
2334.78
1272.18
107.94
7.20
100.85
13.45

<0.0001
<0.0001
0.0011
0.0001
<0.0001
<0.0001
<0.0001
0.0314
<0.0001
0.0080

Significant
Significant
Significant
Significant
Significant
Significant
Significant
Significant
Significant
Significant

Table 5: Coefficient of the model for kerosene oil biodegradation.

J Pet Environ Biotechnol

ISSN: 2157-7463 JPEB, an open access journal

hydrocarbons removal has been clearly shown before in the previous

works of Mohajeri et al. [38]. The quadratic effect of the independent
biostimulating factors on the rate of kerosene removal was significant
but negative.
Figure 2a shows the predicted versus actual plot of kerosene oil
biodegradation. Actual values were determined for a particular run,
and the predicted values were calculated from the approximating
function used for the model. Figure 2b shows the studentized residuals
and normal per cent probability plot. Residual shows the difference
between the observed value of a response measurement and the value
that is fitted under the theorized model. Small residual values indicate
that model prediction is accurate. The Cooks distance and studentized
residuals illustrate the normal distribution and constant variance of
the residuals, the goodness of fit, linearity of the fitted model, and the
independence. Figure 2c shows Cooks distance plot; according to this
plot there were no points that were potentially powerful due to their
location in the factor.

Interaction among factors that influence kerosene oil removal

Table 5 showed that kerosene oil removal was influenced positively
by the interaction of NPK fertilizer (A) and Tween 80 (B); NPK
fertilizer (A) and hydrogen peroxide (C) and interaction of Tween 80
(B) and hydrogen peroxide (C). The graphical representation of the
response shown in Figures 3a c helped to visualize the effect of NPK
fertilizer (A), Tween 80 (B) and hydrogen peroxide (C) on removal of
kerosene. The effect of interaction of NPK fertilizer and Tween 80 on
kerosene bioremediation is illustrated in Figure 3a. It is seen that higher
rate of kerosene removal was attained with higher surfactant (Tween
80) concentration and relatively high amount of NPK fertilizer. The
maximum degradation yield of kerosene (74.95 %) was obtained with
10 mg/l of Tween 80 surfactant and 4 g/l (or 0.02 g/kg) of NPK fertilizer
at a fixed hydrogen peroxide concentration of 1.0 g/l. This may be due
to better bioavailability of substrate for the intrinsic microorganisms.
However, availability of hydrocarbon-utilizing microorganism is a key
issue in crude oil bioremediation [53]. Further increase in the NPK
fertilizer concentration (> 4.0 g/l) resulted in a significant decrease in
the percent kerosene oil removal.
Figure 3b shows the 3D response surface plot of the interaction effect
between NPK fertilizer and hydrogen peroxide (H2O2) concentrations.
This plot demonstrates that both NPK fertilizer and hydrogen peroxide
have the best interaction performance at optimum concentrations. It
could be seen that a higher percent kerosene oil removal was obtained
at a higher hydrogen peroxide concentration with relatively high
amount of NPK fertilizer. This three dimensional plot explains that
both NPK fertilizer and hydrogen peroxide has individual impact
on kerosene oil removal as the individual coefficient of both NPK
fertilizer and hydrogen peroxide is positive and their interaction effect
is positive. However, the impact of NPK fertilizer is more than that of
hydrogen peroxide concentration as the individual coefficient value is
higher for NPK fertilizer (1.45) with lower P-value (<0.0001) than for
hydrogen peroxide (0.55) with higher P-value (0.0011). The maximum
bioremediation yield (75.07%) was achieved with around 1.0 g/l of
hydrogen peroxide and 4 g (0.02 g/kg) of NPK fertilizer. Further
increase in the amount of NPK fertilizer (> 4.0 g/l) tends to decrease
the percent kerosene oil removal. This suggests that hydrogen peroxide
can be increased and that of NPK concentration has to be decreased for
higher percent of kerosene removal.

Volume 3 Issue 3 1000120

Citation: Agarry SE, Owabor CN, Yusuf RO (2012) Enhanced Bioremediation of Soil Artificially Contaminated with Kerosene: Optimization of
Biostimulation Agents through Statistical Experimental Design. J Pet Environ Biotechnol 3:120. doi:10.4172/2157-7463.1000120

Page 6 of 8

A)

B)

C)

1.12

75.00

99

0.84

90

Cooks Distance

72.49

80
70

Predicted

Normal % Probabillity

95

50
30
20

69.98

10

0.28

67.47

0.56

0.00

64.95
-1.93

-0.97

0.00

0.97

1.93

64.95

67.47

69.98

Studentized Residuals

72.49

75.00

Actual

11

13

15

17

Run Number

Figure 2: (a) Normal plot of residuals plot of kerosene oil bioremediation, (b) Predicted versus actual plot of kerosene oil bioremediation, (c) Cooks distance plot of
kerosene oil bioremediation.

74.954

69.9539
67.4538
64.9537

15.00

73.0745

Kerosene Oil Removal (%)

Kerosene Oil Removal (%)

Kerosene Oil Removal (%)

74.9211

75.0677

72.4539

71.0814
69.0882
67.095

12.50

5.00
10.00

B: Tween 80

4.00
7.50

3.00
5.00

A: NPK fertilizer

2.00

(a) NPK and Tween 80

72.3174
71.0156
69.7137

1.50

1.50

6.00

73.6193

6.00

1.25
1.00

C: Hydrogen Peroxide

4.00
0.75

3.00
0.50

12.50
1.00

C: Hydrogen Peroxide

A: NPK fertilizer

10.00
0.75

7.50
0.50

2.00

(b) NPK and Hydrogen Peroxide

15.00

1.25

5.00

B: Tween 80

5.00

(c) Tween 80 and Hydrogen Peroxide

Figure 3: Response surface 3D plots indicating interaction effects of factors (a) NPK and Tween 80, (b) NPK and Hydrogen Peroxide, (c) Tween 80 and Hydrogen
Peroxide.

Figure 3c shows the response surface 3D plot of the effect of

interaction between Tween 80 and hydrogen peroxide concentrations.
Higher rate of kerosene oil removal was observed with increase in
hydrogen peroxide and Tween 80 concentration due to positive
interaction effect. It is evident that due to dominating interaction effects
of hydrogen peroxide, higher levels of this variable give higher yields of
kerosene oil removal. Maximum kerosene oil removal (74.92%) was
obtained with about 1.0 g/l of hydrogen peroxide and 10 mg/l of Tween
80. Further increase in Tween 80 concentration (> 10.0 mg/l) tends to
decrease the percent kerosene oil removal.

Factor plot
The factor effect function plot (Figure 4) was used to assess the
effect of each factor graphically. From the trace plot as shown in Figure
4, it can be seen that each of the three variables used in the present
study has its individual effect on kerosene removal by the intrinsic
microbial populations in the soil. Gradual increase in NPK fertilizer,
Tween 80 and hydrogen peroxide concentrations from low level
(coded value 1) to a higher level (coded value +1) resulted in both
increase and decrease of kerosene oil degradation. Moreover, it is also
J Pet Environ Biotechnol
ISSN: 2157-7463 JPEB, an open access journal

to be noted from Figure 4 that over the range of -1 (2 g) to +1 (6 g) of

NPK fertilizer, the kerosene degradation change in a wide range, which
was also the case for Tween 80. However, for hydrogen peroxide the
kerosene oil removal did not change over a wide range. This clearly
indicates that keeping hydrogen peroxide at the optimum level, a
change in NPK fertilizer and Tween 80 concentrations will respectively
affect the process more severely than done otherwise.

Optimization and validation

Numerical optimization technique based on desirability function
was carried out to determine the workable optimum conditions for
the kerosene oil bioremediation process. In order to provide an ideal
case for biodegradation, the goal for NPK fertilizer, Tween 80 and
hydrogen peroxide was set in range based upon the requirements of the
oil bioremediation and kerosene oil removal was set on maximize. The
predicted optimum (uncoded) values of NPK fertilizer, Tween 80 and
hydrogen peroxide were found to be 4.30 g (equivalent to 0.0215 g/
kg), 10.03 mg/l and 1.13 g/l, respectively, to achieve 75.06% maximum
kerosene oil removal; while desirability for the predicted optimum
values was 1.000 (Figure 5). Nevertheless, validation experiment was

Volume 3 Issue 3 1000120

Citation: Agarry SE, Owabor CN, Yusuf RO (2012) Enhanced Bioremediation of Soil Artificially Contaminated with Kerosene: Optimization of
Biostimulation Agents through Statistical Experimental Design. J Pet Environ Biotechnol 3:120. doi:10.4172/2157-7463.1000120

Page 7 of 8

Kerosene Removal (%)

75

C
C

72.515

B
A

B
70.03

A
67.545

and four variables on the bioremediation of petroleum-contaminated

soil showed that the hydrocarbon removal rate was around 80%.

Conclusions
Significant variations in the kerosene oil biodegradation pattern
were observed with respect to nutrient (NPK fertilizer), surfactant
(Tween 80) and oxygen contents (Hydrogen peroxide). The results of the
present study indicate that biostimulation of kerosene oil contaminated
soil resulted in better petroleum hydrocarbon degradation. Response
Surface Methodology (RSM) is a reliable and powerful tool for
modeling and optimizing of kerosene oil bioremediation processes, in
the optimum conditions petroleum hydrocarbons were degraded up to
73.95% in soil.
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65.06
-1.000

-0.500

0.000

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1.000

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Desirability

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Volume 3 Issue 3 1000120

Citation: Agarry SE, Owabor CN, Yusuf RO (2012) Enhanced Bioremediation of Soil Artificially Contaminated with Kerosene: Optimization of
Biostimulation Agents through Statistical Experimental Design. J Pet Environ Biotechnol 3:120. doi:10.4172/2157-7463.1000120

Page 8 of 8
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