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Studies on Biodegradation of Kerosene in Soil under Different

Bioremediation Strategies

S. E. Agarrya; C. N. Owaborb; R. O. Yusufa

a
Biochemical Engineering Research Laboratory, Department of Chemical Engineering, Ladoke
Akintola University of Technology, Ogbomoso, Nigeria b Department of Chemical Engineering,
University of Benin, Benin-City, Nigeria
Online publication date: 21 July 2010

To cite this Article Agarry, S. E. , Owabor, C. N. and Yusuf, R. O.(2010) 'Studies on Biodegradation of Kerosene in Soil

under Different Bioremediation Strategies', Bioremediation Journal, 14: 3, 135 141

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Bioremediation Journal, 14(3):135141, 2010

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ISSN: 1088-9868
DOI: 10.1080/10889868.2010.495364

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Studies on Biodegradation of Kerosene in

Soil under Different Bioremediation
Strategies
S. E. Agarry,1
C. N. Owabor,2 and
R. O. Yusuf1
1
Biochemical Engineering
Research Laboratory,
Department of Chemical
Engineering, Ladoke Akintola
University of Technology,
Ogbomoso, Nigeria
2
Department of Chemical
Engineering, University of
Benin, Benin-City, Nigeria

Address correspondence to S. E.
Agarry, Biochemical Engineering
Research Laboratory, Department of
Chemical Engineering, Ladoke
Akintola University of Technology,
Ogbomoso, Nigeria. E. mail:
sam agarry@yahoo.com.

ABSTRACT The effectiveness of bioremediation is often a function of the

microbial population and how they can be enriched and maintained in an
environment. Strategies for inexpensive in situ bioremediation of soil contaminated with petroleum hydrocarbons include stimulation of the indigenous
microorganisms by introduction of nutrients (biostimulation) and/or through
inoculation of an enriched mixed microbial culture into soil (bioaugmentation). To demonstrate the potential use of bioremediation in soil contaminated
with kerosene, a laboratory study with the objective of evaluating and comparing the effects of bioattenuation, biostimulation, bioaugmentation, and combined biostimulation and bioaugmentation was performed. The present study
dealt with the biodegradation of kerosene in soil under different bioremediation treatment strategies: bioattenuation, biostimulation, bioaugmentation,
and combined biostimulation and bioaugmentation, respectively. Each treatment strategy contained 10% (w/w) kerosene in soil as a sole source of carbon
and energy. After 5 weeks of remediation, the results revealed that bioattenuation, bioaugmentation, biostimulation, and combined biostimulation and
bioaugmentation exhibited 44.1%, 67.8%, 83.1%, and 87.3% kerosene degradation, respectively. Also, the total hydrocarbon-degrading bacteria (THDB)
count in all the treatments increased with time up till the second week after
which it decreased. The highest bacterial growth was observed for combined
biostimulation and bioaugmentation treatment strategy. A first-order kinetic
model equation was fitted to the biodegradation data to further evaluate the
rate of biodegradation and the results showed that the specific degradation
rate constant (k) value was comparatively higher for combined biostimulation and bioaugmentation treatment strategy than the values for other treatments. Therefore, value of the kinetic parameter showed that the degree of
effectiveness of these bioremediation strategies in the clean up of soil contaminated with kerosene is in the following order: bioattenuation < bioaugmentation < biostimulation < combined biostimulation and bioaugmentation. Conclusively, the present work has defined combined biostimulation
and bioaugmentation treatment strategy requirements for kerosene oil degradation and thus opened an avenue for its remediation from contaminated
soil.
135

KEYWORDS bacteria, bioaugmentation, biodegradation,

bioremediation, biostimulation, kerosene

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INTRODUCTION
The worldwide high demand for petroleum and associated products as a source of energy has resulted in
increased oil exploration, production, and refining, and
has consequently led to a high level of environmental
pollution. Oil spills due to blow outs, leakage from
underground storage tanks, tanker accidents, sabotage,
and accidental rupture of pipelines as well as dumping
of waste petroleum products introduce nonorganic, carcinogenic, and growth-inhibiting chemicals present in
the crude oil and their toxicity to microorganism and
man is well known (Atlas and Bartha, 1973; Okpokwasili and Odokuma, 1990). Also, it results in significant decline in the quality of soil and makes it unfit
for use (Shabir et al., 2008) as well as affects plants and
animal (Plohl et al., 2002).
Crude oil is an extremely complex mixture of
aliphatic and aromatic hydrocarbons, including volatile
components of gasoline, petrol, kerosene, diesel, lubricant oil, and solid asphaltene residues; however, the
kerosene fraction poses the greatest pollution problem
(Solano-Serena et al., 2000; Shabir et al., 2008).
Among several remediation technologies available
for petroleum hydrocarbons removal from the soil
and groundwater, bioremediation technology is gaining prominence due to its simplicity, environmental
friendliness, higher efficiency, and cost-effectiveness in
comparison to other technologies (Alexander, 1994;
Mariano et al., 2007). This bioremediation technology
relies on the natural ability of microorganisms to carry
out the degradation or mineralization of petroleum hydrocarbons (or organic chemicals) to carbon dioxide
and water (Duarte da Cunha and Leite, 2000; Mariano et al., 2007). The actual mechanism that breaks
down these petroleum products is biodegradation mediated by microorganisms (Atlas, 1981; Margesin and
Schinner, 2001).
The effectiveness of bioremediation is often a function of the microbial population and how they can be
enriched and maintained in an environment (Mehrasbi
et al., 2003). The strategies to accelerate the biodegradation of hydrocarbons in soil include biostimulation and
bioaugmentation. Biostimulation involves the addition
of nutrients, electron acceptors, or electron donors to
S. E. Agarry et al.

increase the number or stimulate the activity of indigenous biodegradative microorganisms (Widada et al.,
2002). Bioaugmentation involves the addition or inoculation of indigenous or nonindigenous laboratory
grown microorganisms capable of biodegrading the target pollutant or contaminant (Widada et al., 2002).
Generally, crude oil as well as other commercial hydrocarbons could be considered extensively biodegradable in soils (Shabir et al., 2008); however, differences
in the extent of biodegradation depending on soil and
hydrocarbon source type, concentration of total hydrocarbons, and oxygen and nutrient availability (Shabir
et al., 2008) have been reported in the literature (Bossert
and Compeau, 1995; Moran and Watkinson, 1989;
Shabir et al., 2008). Several studies have been conducted on microbial degradation of petroleum hydrocarbons as obtained in the literature but much work has
not been done on the biodegradation of some commercial petroleum products such as kerosene (Shabir
et al., 2008). Also, the evaluation of natural attenuation,
biostimulation, and bioaugmentation in the bioremediation of soil contaminated with diesel oil has been
carried out (Bento et al., 2003). However, there is little
or no information in the literature on the bioremediation of soil contaminated with kerosene using different
bioremediation methods so as to evaluate and compare
their performance. Therefore, an effort has been made
to remediate soil contaminated with kerosene by using
different bioremediation strategies.
The purpose of the present study was to investigate
and evaluate the effects of bioattenuation, biostimulation, bioaugmentation, and combined biostimulation
and bioaugmentation on kerosene degradation through
the application of a first-order kinetic model equation.

MATERIALS AND METHODS

Kerosene, Fertilizer, and Chemicals
Kerosene purchased from a petroleum filling station was used as sole carbon and energy source.
All the chemicals used were of analytical grade. The
NPK chemical fertilizer purchased from a local agrochemical store was used as a biostimulating agent.

Soil Sample Collection and

Characterization
Soil sample was randomly collected with a Dutch
auger at a depth of 15 cm from an agricultural field
136

TABLE 1 Physicochemical and Microbiological Properties of

Soil

Soil parameters
Moisture content (%)
Total nitrogen (%)
Available phosphorus (%)
Potassium (%)
Total organic carbon (%)
pH
Total hydrocarbon-degrading bacteria
(THDB) (CFUg1 )

Value
5.95 0.05
0.25 0.04
0.12 0.02
0.31 0.05
1.21 0.03
5.9 0.2
3.7 105

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Note. Each value is a mean of three replicates and indicates standard

deviation among them.

in Ogbomoso, Nigeria. The soil samples were homogenized, dried, sieved through a 2-mm mesh screen,
and stored in a polythene bag at room temperature
in the laboratory. The soil was characterized for physicochemical and microbial parameters. Soil pH was determined according to the modified method of McLean
(1982); total organic carbon was determined by the
modified wet combustion method (Nelson and Sommers, 1982) and total nitrogen was determined by the
semi-micro-Kjeldhal method (Bremner and Mulvaney,
1982). Available phosphorus was determined by Brays
No. 1 method (Olsen and Sommers, 1982) and moisture content was determined by the dry weight method.
The total hydrocarbon degrading bacteria (THDB) populations was determined by the vapor phase transfer
method (Amanchuckwu et al., 1989).
The soil characterization showed that the soil did
not fulfill the nutrient (NPK) requirements for an efficient biodegradation process (Table 1). Therefore, these
elements were added in the form of NPK chemical fertilizer (20:10:10) to provide the proper nutrients for the
biodegradation process.

Binary Mixed Culture (Inoculum)

Preparation
Nonindigenous oil-degrading bacteria strains of
Pseudomonas aeruginosa and Pseudomonas putida were
obtained from the Department of Pure and Applied
Biology, Ladoke Akintola University of Technology,
Ogbomoso, Nigeria. These were subcultured on
separate nutrient agar McCarty bottles and incubated
at 37 C for 24 h. Single colonies of these organisms
were transferred separately into 250-ml Erlenmeyer
flasks containing 100 ml of the nutrient broth medium
137

(0.8%) made up of composition: yeast extract 2.0 g/L,

peptone 5.0 g/L, NaCl 5.0 g/L, and agar 15.0 g/L, incubated at 37 C for 48 h in a New Brunswick gyratory
shaker (G-25 model; New Jersey, USA), and agitated at
120 rpm. Aliquots (50 ml) of each culture medium were
centrifuged at 4900 g for 15 min. The cell pellets were
washed and resuspended in autoclaved normal saline
(0.89% NaCl in distilled water). Ten milliliter of each
cell suspension was mixed in a sterilized screw-capped
test tube and this was the mixed bacterial culture.

Enrichment of Binary Mixed Bacterial

Culture
One milliliter of the cell suspension (mixed bacterial
culture) was transferred to mineral salt medium containing per liter: K2 HPO4 1.0 g, KH2 PO4 1.0 g, NH4 NO3
1.0 g, MgSO4 0.2 g, and CaCl2 0.02 g and kerosene
(3% w/v) in a 250-ml Erlenmeyer flask and incubated
for 48 h at 37 C in a gyratory shaker agitated at a speed
of 120 rpm. After 48 h of growth, in the same manner,
1 ml of the culture was transferred to serially increasing
concentration of kerosene up to 10% (w/v). Cells were
recovered by centrifugation and resuspended in normal
saline to set an OD of 0.7 at 660 nm. This inoculum
(10% w/v) was the one used for bioaugmentation to
study the biodegradation of kerosene in soil.

Experimental Design and Soil

Treatment
Four earthen pots were prepared for each treatment,
designated as bioattenuation (treatment B), bioaugmentation (treatment C), biostimulation (treatment D), and
combined biostimulation and bioaugmentation (treatment E). Another four earthen pots were prepared for
control experiment designated as B1, C1, D1, and E1
for treatment B, treatment C, treatment D, and treatment E, respectively. Each earthen pot contained 190 g
of soil, contaminated and mixed well with 19 g of
kerosene (10% w/w). The pot under treatment C was
amended with 40 ml of inoculum (3.2 105 colonyforming units [CFU]g1 ), and the pot under treatment D was amended with 2.56 g of NPK fertilizer,
respectively. The pot under treatment E was amended
with both 40 ml of inoculum and 2.56 g of NPK fertilizer. The pot under treatment B was not amended
with either NPK fertilizer and/or inoculum. The control pots were not amended with either NPK fertilizer
Biodegradation of Kerosene

in the samples were determined by using the stored calibration graph in the software of the equipment on a
reference.

Kinetics of Degradation
The rate of petroleum hydrocarbon biodegradation
was measured by the application of first-order kinetic
model equation (Equation 1) to the biodegradation
data, which has generally been used for biodegradation
of petroleum hydrocarbons in soil (Guerin, 1999; Roncevic et al., 2005; Abassi and Shquirat, 2008; Adesodun
and Mbagwu, 2008).
S = SoK t

(1)

Taking the natural logarithm of Equation 1

Estimation of Residual Kerosene

ln(S/S0 ) = kt

The residual kerosene was determined in accordance

with American Society for Test and Materials (ASTMD5369). Fourier transform infrared spectrophotometer
(FTIR; Genesis series) was used for the test. Five grams
of soil sample was weighed into 250-ml Erlenmeyer
conical flask and 50 ml of carbon tetrachloride solvent added to extract the oil in the soil. After shaking vigorously, the mixture was allowed to stand for
10 min and separated using separating funnel. It was
then filtered through Whatman No. 1 filter paper. The
equipment was calibrated with isooctane/acetone in
carbon tetrachloride. Residual kerosene concentrations

Where S, So , k, and t are the initial petroleum hydrocarbon concentration, final petroleum hydrocarbon
concentration, specific degradation rate constant, and
time, respectively.

(2)

RESULTS AND DISCUSSION

Figure 1 shows the degradation profile of kerosene
as a function of time for each bioremediation treatment strategy, bioattenuation, bioaugmentation, biostimulation, and combined biostimulation and bioaugmentation. From Figure 1, it is revealed that kerosene

12

Kerosene concentration (w/w)

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or inoculum. All the control pots with its contents

were sterilized three times by autoclaving at 121 C for
15 min. All the eight earthen pots with its contents
were incubated at room temperature (28 C 2 C) for
5 weeks.
The study was carried out during the rainy season between the month of June and July when the climate is
cool and the temperature is usually not high. The water
(moisture) content of soil in each pot was adjusted every
week by addition of sterile distilled water to a moisture
holding capacity of 50%. In order to avoid anaerobic
conditions, contents of the pots were aerated by mixing every 3 days. Samples were taken every week and
analyzed for residual kerosene and total hydrocarbon
degrading bacteria, respectively. The experiments were
carried out in triplicates.

10

Bioattenuation

Bioaugmentation

Biostimulation

Combined biostimulation and

bioaugmentation
Control

2
0
0

10

20

30

40

Remediation time (days)

FIGURE 1 Kerosene degradation as a function of time.
S. E. Agarry et al.

138

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degradation began during the first week of remediation in all the treatments and slowly continued up to 5 weeks. The concentration of kerosene
(10% w/w) was reduced to 5.6%, 3.2%, 1.7%, and
1.3% in 5 weeks of remediation and correspondingly
44.1%, 67.8%, 83.1%, and 87.3% kerosene reduction was achieved under bioattenuation, bioaugmentation, biostimulation, and combined biosimulation
and bioaugmentation treatments, respectively. Thus,
combined biostimulation and bioaugmentation treatment strategy (which have not been reported to have
been used for kerosene removal) showed relatively more
kerosene reduction than other treatments during the
whole period of remediation.
Moreover, it may be seen that during the remediation period, chemical oxidation processes and
metabolic activities including biodegradation were enhanced by nutrients; thus, kerosene degradation was
higher in biostimulation treatment than bioattenuation treatment. A similar observation has been reported
(Hadhrami et al., 1997; Shabir et al., 2008). In contrast,
bioattenuation treatment has been reported to be better
than biostimulation in the bioremediation of soil contaminated with diesel oil (Bento et al., 2003). Therefore, during the biodegradation of kerosene in soil,
addition of nutrient fertilizer and bacterial inoculum
individually resulted in an effective bioremediation response. This is in agreement with earlier research reports
(Bourquin, 1996; Raza et al., 2006, 2007; Shabir et al.,
2008). However, among the individual methods of natural attenuation, biostimulation, and bioaugmentation
that were used for the remediation of a soil contaminated by diesel oil, bioaugmentation was reported to be
the best method (Bento et al., 2003). A small reduction
in kerosene concentration was observed in control setup (Figure 1). This reduction may be due to combined
effects of abiotic degradation of kerosene and evaporation losses. A similar observation has been reported
(Shabir et al., 2008).
Fitting the biodegradation data obtained for each
soil treatment to first-order kinetic model using the linear regression routine of MATLAB 7.0 software package
made possible further evaluation and comparison of the
applicability of the various bioremediation strategies.
The parameter k of the model, being the specific degradation rate constants for biodegradation of kerosene in
the soil, was determined for soil treatment by bioattenuation, bioaugmentation, biostimulation, and combined
biostimulation and bioaugmentation, respectively. The
139

TABLE 2 Specific Degradation Rate Constant (k) and Correlation Coefficient (R2 )

Soil treatment
Bioattenuation
Bioaugmentation
Biostimulation
Combined biostimulation and
bioaugmentation

k (day1 )

R2

0.0184
0.0329
0.0503
0.0597

.9625
.9991
.9992
.9967

results are presented in Table 2. The results indicate

that the k value (0.0597 day1 ) for the biodegradation of kerosene in soil under combined biostimulation
and bioaugmentation treatment strategy was comparatively higher than the value for other treatments. The
soil treatment under bioattenuation had the smallest
k value (0.0184 day1 ). Therefore, value of the kinetic
parameter showed that the degree of effectiveness of
these bioremediation strategies in the clean up of soil
contaminated with kerosene is in the following order:
bioattenuation < bioaugmentation < biostimulation
< combined biostimulation and bioaugmentation.
Figure 2 shows the growth profile of the hydrocarbon degrading bacterial population in the soil. An
initial hydrocarbon-degrading bacterial population of
about 4.1 105 , 5.5 105 , 5.0 105 , and 5.7
105 CFUg1 of soil was observed respectively at the
start of remediation for bioattenuation, bioaugmentation, biostimulation, and combined biostimulation
and bioaugmentation treatments, as shown in Figure 2.
This indicated that in the soil, the addition of preselected bacteria (bioaugmentation) and nutrients (biostimulation) resulted in the highest number of bacteria
at the beginning of remediation compared to bioattenuation (or natural attenuation). The lower bacteria
population in bioattenuation treatment may be due to
the presence of low nutrients and the disturbance of
nutrient ratio especially of NPK that is needed in the
correct ratio for bacteria growth. As seen from Figure
2, a maximum bacterial population of 7.6 105 , 7.0
105 , 8.6 105 , and 5.8 105 CFUg1 of soil was
respectively obtained under bioaugmentation, biostimulation, and combined biostimulation and bioaugmentation treatment strategies at the end of the second
week of remediation, and after that, the population
started to decline. A similar observation has been reported (Mehrasbi et al., 2003; Ayotamuno et al., 2007;
Shabir et al., 2008). The decline in bacterial population
as observed for bioaugmentation, biostimulation, and
Biodegradation of Kerosene

10
Bioattenuation

8
Bioaugmentation

Bacterial count (x10 cfug

-1)

Biostimulation

5
4
3

Combined biostimulation
and bioaugmentation

Control

1
0
0

10
20
30
Remediation time (days)

40

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FIGURE 2 Bacterial growth on kerosene oil.

combined biostimulation and bioaugmentation treatments after the second week of remediation may be
due to reduction in carbon and nutrient source (i.e.,
carbon and nutrient limitation). However, no bacterial growth was observed in the control soil medium
(Figure 2).

CONCLUSION
From this present study, it can be concluded that
the rate of biodegradation of kerosene in soil could be
enhanced by the addition of nutrients and inoculum,
respectively. The soil treatment under combined biostimulation and bioaugmentation exhibited the highest degree of degradation and the soil treatment under bioattenuation the least degradation. Thus, the use
of biostimulation and bioaugmentation to enhance
kerosene degradation in the soil could be one of the
severally sought bioremediation strategies of remediating natural ecosystem (environment) contaminated
with petroleum hydrocarbons.

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Biodegradation of Kerosene