Professional Documents
Culture Documents
Biotechnology
Research Article
Open Access
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.
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
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
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.
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.
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 (
2.0
4.0
6.0
Tween 80 (B )
5.0
10
15
Hydrogen Peroxide ( C)
0.5
1.0
1.5
Tween 80 (B)
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.
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
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
-
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].
40
30
20
10
0
1
Natural Bioattenuation
10
11
12
13
14
15
16
17
Experimental Run
Enhanced Bioremediation
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
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
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
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
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
15.00
1.25
5.00
B: Tween 80
5.00
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.
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
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
75
C
C
72.515
B
A
B
70.03
A
67.545
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.
References
1. Wikipedia (2005) Kerosene. The free encyclopedia.
65.06
-1.000
-0.500
0.000
0.500
1.000
2. Gouda MK, Omar SH, Nour Eldin HM, Chekroud ZA (2008) Bioremediation
of kerosene II: a case study in contaminated clay (Laboratory and field-scale
microcosms). World J Microbiol Biotechnol 24: 1451-1460.
3. Irwin RJ, Mouwerik MV, Stevens L, Seese MD, BashamW (1997) Environmental
Contaminants Encyclopedia Kerosene Entry. National Park Service Colorado.
1.000
0.752
Desirability
0.505
0.257
0.009
15.00
6.00
12.50
5.00
10.00
B: T w e e n 8 0
4.00
7.50
3.00
5.00
2.00
A: N P K fertilizer
9. Atlas RM, Bartha R (1973) Fate and effects of polluting petroleum in the marine
environment. Residue Rev 49: 49-85.
10. Kao CM, Chen CY, Chen SC, Chien HY, Chen YL (2008) Application of in
situ biosparging to remediate a petroleum- hydrocarbon spill site: Field and
microbial evaluation. Chemosphere 70:14921499.
11. Jobson A, Mclauglin M, Cook FD, West lake DW (1974) Effect of amendments
on the microbial utilization of oil applied to soil. Appl Microbiol 27: 166 -171.
12. Lee SH, Lee S, Kim DY, Kim JG (2007) Degradation characteristics of waste
lubricants under different nutrient condition. J Hazard Mater 143: 65-72.
13. Coulon F, Pelletier E, Gourhant L, Delille D (2005) Effects of nutrient and
temperature on degradation of petroleum hydrocarbons in contaminated subAntarctic soil. Chemosphere 58: 1439-1448.
14. Ayotamuno MJ, Kogbara RB, Ogaj SOT, Probert SD (2006) Bioremediation of
a crude-oil polluted agricultural-soil at Port Harcourt, Nigeria. Appl Energy 83:
1249-1257.
15. Agarry SE, Owabor CN, Yusuf RO (2010) Bioremediation of soil artificially
contaminated with petroleum hydrocarbon mixtures: Evaluation of the use of
animal manure and chemical fertilizer. Bioremediat J 14: 189195.
16. Arienzo M (2000) Degradation of 2, 4, 6-trinitrotoluene in water and soil slurry
utilizing a calcium peroxide compound. Chemosphere 40: 331-337.
17. Parreira AG, Ttola MR, Jham GN, Da Silva SL, Borges AC (2011) Microbial
biodegradation of aromatic compounds in a soil contaminated with gasohol.
British Biotechnology Journal 1: 18-28.
18. Tsai TT, Kao CM, Surampalli RY, Chien HY (2009) Enhanced Bioremediation
of Fuel-Oil Contaminated Soils: Laboratory Feasibility Study. J Environ Eng
135: 845-853.
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
19. Yeh CK, Wu HM, Chen TC (2003) Chemical oxidation of chlorinated non
aqueous phase liquids by hydrogen peroxide in natural sand systems. J Hazard
Mater 96: 2951.
20. Ferguson SH, Woinarski AZ, Snape I., Morris CE, Revill AT (2004) A field
trial of in situ chemical oxidation to remediate long-term diesel contaminated
Antarctic soil. Cold Regions Sci Technol 40: 4760.
21. Lodha B, Chaudhari S (2007) Optimization of Fenton-biological treatment
scheme for the treatment of aqueous dye solutions. J Hazard Mater 148: 459
466.
22. Mulligan CN, Yong RN, Gibbs BF (2001) Surfactant enhanced remediation of
contaminated soil: A review. Engineering Geology 60: 371380.
23. Franzetti A, Di Gennaro P, Bestetti G, Lasagni M, Pitea D, et al. (2008)
Selection of surfactants for enhancing diesel hydrocarbons-contaminated
media bioremediation. J Hazard Mater 152: 13091316.
24. Ahmadi M, Vahabzadeh F, Bonakdarpour B, Mofarrah E, Mehranian M (2005)
Application of the central composite design and response surface methodology
to the advanced treatment of olive oil processing wastewater using Fentons
peroxidation. J Hazard Mater 123: 187-195.
25. Rigas F, Papadopoulou K, Dritsa V, Doulia D (2007) Bioremediation of a soil
contaminated by lindane utilizing the fungus Ganoderma australe via response
surface methodology. J Hazard Mater 140: 325-332.
26. Huang L, Ma T, Li D, Liang FL, Liu RL, et al. (2008) Optimization of nutrient
component for diesel oil degradation by Rhodococcus erythropolis. Mar Pollut
Bull 56:1714-1718.
28. Piehler MF, Swistak JG, Pinckney JL, Paerl HW (1999) Stimulation of Diesel
Fuel Biodegradation by Indigenous Nitrogen Fixing Bacterial Consortia. Microb
Ecol 38: 6978.
49. Joo HS, Ndegwa PM, Shoda M, Phae CG (2008) Bioremediation of oilcontaminated soil using Candida catenulata and food waste. Environ Pollut
156: 891896.
29. Rhykerd RL, Crews B, McInnes KJ, Weaver RW (1999) Impact of bulking
agents, forced aeration and tillage on remediation of oil-contaminated soil.
Bioresour Technol 67: 279285.
Special features: