Journal of Environmental Management 180 (2016) 197e201

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Journal of Environmental Management

journal homepage: www.elsevier.com/locate/jenvman

Research article

The effect of soil type on the bioremediation of petroleum

contaminated soils
Ali Haghollahi a, Mohammad Hassan Fazaelipoor a, b, *, Mahin Schaffie a, b
a
Mineral Industries Research Center, Shahid Bahonar University of Kerman, Iran
b
Department of Chemical Engineering, Faculty of Engineering, Shahid Bahonar University of Kerman, Iran

a r t i c l e i n f o a b s t r a c t

Article history: In this research the bioremediation of four different types of contaminated soils was monitored as a
Received 19 February 2016 function of time and moisture content. The soils were categorized as sandy soil containing 100% sand
Received in revised form (type I), clay soil containing more than 95% clay (type II), coarse grained soil containing 68% gravel and
13 May 2016
32% sand (type III), and coarse grained with high clay content containing 40% gravel, 20% sand, and 40%
Accepted 14 May 2016
Available online 1 June 2016
clay (type IV). The initially clean soils were contaminated with gasoil to the concentration of 100 g/kg,
and left on the floor for the evaporation of light hydrocarbons. A full factorial experimental design with
soil type (four levels), and moisture content (10 and 20%) as the factors was employed. The soils were
Keywords:
Total petroleum hydrocarbon (TPH)
inoculated with petroleum degrading microorganisms. Soil samples were taken on days 90, 180, and 270,
Bioremediation and the residual total petroleum hydrocarbon (TPH) was extracted using soxhlet apparatus. The moisture
Moisture content of the soils was kept almost constant during the process by intermittent addition of water. The
Soil type results showed that the efficiency of bioremediation was affected significantly by the soil type
Sandy soil (Pvalue < 0.05). The removal percentage was the highest (70%) for the sandy soil with the initial TPH
Clay soil content of 69.62 g/kg, and the lowest for the clay soil (23.5%) with the initial TPH content of 69.70 g/kg.
The effect of moisture content on bioremediation was not statistically significant for the investigated
levels. The removal percentage in the clay soil was improved to 57% (within a month) in a separate
experiment by more frequent mixing of the soil, indicating low availability of oxygen as a reason for low
degradation of hydrocarbons in the clay soil.
© 2016 Elsevier Ltd. All rights reserved.

1. Introduction hydrocarbons and their low degradation rates require special

Petroleum hydrocarbons are persistent pollutants in the envi-
ronment. Uncontrolled release of these compounds affects soil,
water, and air negatively (Ulrici, 2008). Soil pollution with hydro-
carbons is caused by the leakage from underground reservoirs,
petroleum refineries, storage facilities, and accidental spillage from
production units and transport pipelines. The presence of petro-
leum hydrocarbons affects physical, physiological and biochemical
properties of soil (Margesin et al., 2003; Head et al., 2006). Plants
are susceptible to oil exposure due to phytotoxic nature of hydro-
carbons, and immobilization of nutrients in the soil (De Jong, 1980;
Udo and Fayemi, 1995). Inherent mutagenic properties of some
* Corresponding author. Department of Chemical Engineering, Faculty of Engi-
neering, Shahid Bahonar University of Kerman, Iran.
E-mail addresses: fazaelipoor@uk.ac.ir, fazaelipoor@yahoo.com
(M.H. Fazaelipoor).
attention to remediate these pollutants (Johnsen et al., 2007).
Various physical, chemical and thermal methods have been
employed to clean up oil contaminated sites (Frick et al., 1999).
More recently, bioremediation has been suggested as a cost effec-
tive and environmental friendly method for soil cleaning. In
bioremediation the capability of biological agents is exploited to
degrade hydrocarbons. A large body of literature exist that suggest
bioremediation as a cost effective and environmental friendly
method of soil cleaning (Kwok and Loh, 2003; Glick, 2003; Zhuang
et al., 2007; Gerhardt et al., 2009; Dindar et al., 2013; Sarma Roy
et al., 2014).
Moisture content of soil, microbial population, nutrient avail-
ability, soil type, salinity, and oxygen transport in soil are among
the factors affecting the process of bioremediation. The moisture
content of soil should be at an optimum range. Low levels of
moisture content decrease microbial activity, while excess water
may create resistance to oxygen transfer and may also produce an
unwanted leachate (Schjønning et al., 2011). In bioremediation the

http://dx.doi.org/10.1016/j.jenvman.2016.05.038
0301-4797/© 2016 Elsevier Ltd. All rights reserved.
198 A. Haghollahi et al. / Journal of Environmental Management 180 (2016) 197e201
Table 1
Bandar Abbas gasoil components.
Water and sediments Ash Total sulfur Density
0.05%Vol 0.01%wt 1%wt 840 kg/m3
moisture content is normally adjusted to a fraction of water holding
capacity of the soil. The optimum value of moisture, however, is a
function of soil type, pore size distribution, and soil texture. The
information in the literature on the optimum value of the moisture
content of soil for the purpose of bioremediation is scarce, and
more investigation is needed to address this case. In general, op-
timum microbial activity is achieved by the maximum water con-
tent that does not restrict oxygen diffusion. Nitrogen, phosphorous,
sulfur and some other nutrients are necessary for microbial growth
and activity. Therefore contaminated soils should contain ample
amounts of these elements for a successful bioremediation process.
As a rule of thumb the ratio of Carbon: Nitrogen: phosphorous
should be 100:10:1 to ensure a balanced medium for microbial
growth in term of nutrients (Prescott et al., 2002). Soil type and
texture can affect bioremediation. Fine grained soils like clay have
low permeability and retard oxygen and nutrients transport in the
soil. Controlling the moisture content in fine grained soils is diffi-
cult due to having small pores and high surface area (Balba et al.,
1998). Clays can also catalyze humic acid formation and protect
organic materials from decomposition within aggregates (Stott and
Martin, 1990). Bioremediation of clays is therefore a challenging
task. The ratio of external to total surface area of soil particles is also
an important factor since only external surfaces are accessible to
microorganism (Kwok and Loh, 2003).
The effect of soil type is among the less investigated factors
affecting bioremediation. The aim of the present study is to deter-
mine the role of soil type and moisture content in the bioremedi-
ation of highly contaminated soils. The effects of soil type and
moisture content have been investigated using a two factor facto-
rial experimental design.
2. Materials and methods
2.1. Soil
Four different types of soil were selected. Sandy soil containing
100% sand (type I), clay soil containing more than 95% clay (type II),
coarse grained soil containing 68% gravel and 32% sand (type III),
and coarse grained soil with high clay content containing 40%
gravel, 20% sand, and 40% clay (type IV). The soils were categorized
based on the Unified Soil Classification System (USCS). Soils types
I,II, and III were obtained from the suburb of Yazd, Iran (31.8972 N,
54.3678 E), and soil type IV was obtained from the suburb of
Kerman, Iran (30.2907 N, 57.0679 E). The soils were stored in
closed buckets at room conditions before the tests. An investigation
Table 2
Composition of the basal mineral medium used for the initial microbial growth.
1
Compound (g L ) CaCl2$7H2O (0.04) MgCl2$7H2O (0.2)
1
Compound (g L ) MnCl2 (0.001) NaMoO4 (0.00175)
Table 3
Removal percentage of TPH in soil after 270 days of bioremediation.
Moisture
Sandy soil Clay soil
10% 63% 23.5%
20% 70% 17%
on the origin of the soils indicated negligible organic content. The
initially clean soils were contaminated with gasoil to the concen-
Compound tration of 100 g/kg, and left on the floor at room temperature. After
C6eC16
evaporation of the light hydrocarbons, the residual gasoil in the
soils was quantified, and the soils were stored for the next step. The
gasoil was obtained from Bandar Abbas refinery (Table 1).
2.2. Design of experiment for bioremediation
A full factorial experimental design was applied to investigate
the effects of soil type, and moisture content on bioremediation.
Soil type in four levels as mentioned above, and moisture content in
two levels of 10 and 20% were investigated.
The experiment contained 8 runs which were performed in
duplicate. 30 kg soil was used for each run. The soils were placed in
buckets.110 g (NH4)2SO4 and 108 g KH2PO4 were added to each
bucket as nutrient supplements. The chemicals were purchased
from Kiankaveh Azma pharmaceutical & chemicals complex Inc.
These amounts resulted in the approximate ratio of 100:1:1 for
Carbon: Nitrogen: phosphorous in soils at the beginning of the
bioremediation process. The amount of (NH4)2SO4 was considered
lower than usual to avoid possible excessive osmotic pressure in the
microenvironments of the soils.
A group of unidentified petroleum degrading microorganisms
were used in this research. The microorganisms were isolated from
a petroleum contaminated soil, previously undergone a bioreme-
diation process. The microorganisms were grown in a basal mineral
medium with the composition presented in Table 2, and with gasoil
as the substrate. 500 mL of this microbe containing medium was
used as the inoculums for each bucket. The moisture content of the
soils was measured on monthly basis and adjusted with tap water.
The soils were blended thoroughly after the addition of water.
Due the low removal percentage of TPH in clay soil, in a com-
plementary experiment, the soil was subjected to more frequent
water adjustment and mixing. In this experiment, two factors were
examined: water content and mixing. Water content was examined
at five levels of 5, 10, 20, 30, and 40%. Thorough mixing of the soil
every 48 h after moisture adjustment was another factor (versus
intact samples during bioremediation). The experiment was
designed as a two factor factorial experiment having 10 runs. The
runs were performed in duplicate. The samples weighed 500 g. For
this experiment the bioremediation continued for one month.
2.3. Quantification of residual TPH in soil
For quantification of residual TPH is soil, samples (2 g) were
taken and dried in an oven at the temperature of 70  C for 5 h. The
TPH content of the soil samples were extracted by Soxhlet appa-
ratus and quantified based on the EPA Method 9071B (EPA, Method
9071B, 1998). A blank test was done to determine the efficiency TPH
K2HPO4 (4.3) KH2PO4 (3.4) (NH4)2SO4 (4) FeSO4 (0.03)
CuSO4 (0.00015) H3BO3 (0.000375) ZnSO4 (0.0017)
Soil Type
Coarse grained soil Coarse grained soil with high clay content
62.5% 65%
57% 66.5%
 A. Haghollahi et al. / Journal of Environmental Management 180 (2016) 197e201 199

Table 4
Analysis of variance for the effects of soil type and moisture on bioremediation.

Source of variation Degree of freedom Sum of squares Mean sum of squares F Pvalue

Soil type 3 5851.69 1950.56 201.35 0.000

Moisture 1 14.06 14.06 1.45 0.263
Soil type- moisture interaction 3 109.69 36.56 3.77 0.059
Error 8 77.50 9.69

extraction from the soil samples. For this purpose 5 g gasoil was
blended with 10 g dried soil, and put the sample in an oven at 70  C 100

for 24 h for the evaporation of light hydrocarbons. The sample was 80 10% Moisture Con. 20% Moisture Con.

TPH Removal(%)
then weighed to determine the amount of residual gasoil in the soil.
60
Subsequently the sample was subjected to extraction and quanti-
fication by the EPA Method 9071B. This blank test verified that 40

more than 95% of the residual gasoil in the soil could be extracted. 20

0
2.4. GC (Gas chromatograph) and FTIR (Fourier Transform Infrared 0 60 120 180 240 300

spectroscopy) analysis Time (Day)

Fig. 1. TPH removal in sandy soil (type I) as a function of time. Each point represents
The extracted TPH from the sandy soil was analyzed by FTIR and the average value of duplicate samples. Initial TPH 69.625 g/kg.
GC to monitor the changes in the composition of the TPH qualita-
tively. For GC analysis the extracted TPH from the soil sample was
diluted with chloroform and 2 microliter of the resulting solution 100
was injected to the GC. The temperature of injector and detector
were 250  C and 200  C, respectively. The temperature of the col- 80
TPH Removal(%)

umn was 100  C initially, and increased by the rate of 15  C/min to 60

300  C. The column was capillary Varian CP-Sil5CB 10% Moisture Con. 20% Moisture Con.

30 m  0.32 mm  0.10 mm e CP8740. Nitrogen was the carrier 40

gas. For FTIR analysis the extracted oil from the soil was made into 20
pellets using KBr in the ratio of 100:1(100 mg KBr and 1 mg sam-
ple), and spectra were taken in the range of 500e4000 cm 1. 0
0 60 120 180 240 300
Time(Days)
2.5. Statistics
Fig. 2. TPH removal in clay soil (type II) as a function of time. Each point represents the
average value of duplicate samples. Initial TPH 69.700 g/kg.
Analysis of variance (ANOVA) was performed to determine the
significance of the factors using Minitab software.

100
3. Result and discussions
80
TPH Removal(%)

3.1. The effects of soil type and moisture content on the 60

10% Moisture Con. 20% Moisture Con.
bioremediation of gasoil
40

Table 3 shows the results of bioremediation on day 270. The 20

bioremediation was most successful in sandy soil with more than
0
70% degradation of TPH. The removal percentage was relatively low 0 60 120 180 240 300
in clay. In Table 4 the results of the analysis of variance have been Time(Days)
presented. The results shows that soil type significantly affects
bioremediation (Pvalue < 0.05), and the variation in moisture content Fig. 3. TPH removal in coarse grained soil (type III) as a function of time. Each point
represents the average value of duplicate samples. Initial TPH 71.610 g/kg.
does not significantly affect the TPH removal in the tested range. The
removal percentages in other soils were comparable to each other.
The results confirm that the presence of sand in the soil is advan-
tageous in bioremediation. The low bioremediation in clay could be 100
due to inefficient oxygen transfer in the soil. Fine grained clay with
80
TPH Removal(%)

high surface area formed a sticky texture in the presence of water,

blocking efficient oxygen transfer through the soil. Sandy soils on the 60
10% Moisture Con. 20% Moisture Con.
other hand are more porous than clays. Higher porosity allows better 40
oxygen transfer in the soil which is essential to biodegradation of
hydrocarbons. Larger pores provide also enough space for microbial 20

growth. Akbari and Ghoshal (2015) demonstrated that pores smaller 0

than 3 micrometers are not accessible to bacteria. Another reason for 0 60 120 180 240 300
low degradation of hydrocarbons in clay could be strong adsorption Time(Days)

of the pollutants on the surface of soil particles. Fig. 4. TPH removal in coarse grained soil with high clay content (type IV) as a function
Figs. 1e4 show the results of bioremediation for the soils as a of time. Each point represents the average value of duplicate samples. Initial TPH
function of time and moisture content. For the sandy soil (Type I) 69.525 g/kg.
200 A. Haghollahi et al. / Journal of Environmental Management 180 (2016) 197e201

Table 5 frequent mixing the TPH removal increased to 57% in one month.
TPH removal percentage in the clay soil as a function of moisture content and Mixing The removal in unmixed soil samples was lower than the samples
in the complementary experiment.
with mixing. The best result here was for the moisture content of
Moisture content Water addition & mixing Water addition 10%. Higher moisture content negatively affected the TPH removal
5% 35% ± 0.04 30% ± 0.06 in the samples. Table 6 shows the results of the analysis of variance
10% 57% ± 0.03 47% ± 0.08 for this experiment. The results confirm that the moisture content
20% 45% ± 0.04 43% ± 0.03 and blending both significantly affect the TPH removal in clay soils.
30% 35% ± 0.03 30% ± 0.01
Comparing the results of this experiment with the previous one, it
40% 16% ± 0.06 7% ± 0.03
can be concluded that the main reason for low degradation of TPH
in the previous experiment was low availability of oxygen to the
microbes. The large size of the sample (30 kg) made it difficult to
the gasoil residual was 69.63 g/kg at the beginning and decreased
mix it efficiently and this prevented proper exposure of the soil
to 20.6 g/kg on day 270. For the clay soil (Type II) the gasoil residual
aggregates with air. In the latter experiment small size of the
was 69.7 g/kg at the beginning and decreased to 52.9 g/kg on day
samples (500 g) and frequent mixing allowed efficient exposure to
270. For the coarse soil (Type III) the gasoil residual was 71.61 g/kg
the air and improved the rate of biodegradation.
at the beginning and decreased to 26.35 g/kg on day 270, and finally
for the coarse soil with high clay content (Type IV) the gasoil re-
3.2. GC and FTIR analysis
sidual was 69.52 g/kg at the beginning and decreased to22.55 g/kg
on day 270. Overall the results of this part showed that sandy soils
Fig. 5 compares the GC chromatograms for the extracted TPH
are the best candidate for bioremediation while clay soils resist
from the sandy soil on days 0 and 270. The chromatogram for the
bioremediation.
extracted oil on day 270 has lower number of peaks indicating near
The results of the complementary experiment on the clay soil
complete degradation of some components. Other peaks appeared
have been presented in Table 5. The results indicate that by
smaller than the corresponding peaks in the chromatogram

Table 6
Analysis of variance for the effects of moisture content and mixing on the TPH removal in clay.

Source of variation Degree of freedom Sum of squares Mean sum of squares F Pvalue

Moisture 4 3737.69 933.50 116.69 0.000

Mixing 1 192.20 192.20 24.02 0.001
Moisture- mixing interaction 4 42.80 10.70 1.34 0.322
Error 10 80.00 8.00

Fig. 5. Chromatograms of the extracted hydrocarbons from the sandy soil on days 0 (top), and 270 (down).
 A. Haghollahi et al. / Journal of Environmental Management 180 (2016) 197e201
Fig. 6. FTIR Spectra of the extracted hydrocarbons from the sandy soil on days 0 and 270.
obtained for the extracted residual oil on day 0 indicating the
biodegradability of most components of the gasoil. Another point
form comparing the chromatograms is that the total area of the
peaks for the chromatogram of the sample on day 270 is 76.5%
smaller than the total area of the peaks for the chromatogram of the
sample extracted on day 0. This figure is reasonably close to the
removal percentage of TPH (70%) obtained by the gravimetric
method (EPA Method 9071B). The reduction in total area of peaks,
therefore, can be considered as an approximate measure for the
reduction in TPH content of the soils.
The FTIR spectra of the extracted hydrocarbons from the sandy
soil on days 0 and 270 have been presented in Fig. 6. Inspection of
the spectra shows considerable change in the extracted sample
after bioremediation. All spectra show strong absorptions in the
wavelength range of 3000e2840 cm 1. The absorptions arise from
CeH stretching in the alkanes. This indicates that the remaining
hydrocarbons after bioremediation are mainly alkanes. Many of
absorption peaks at lower wavelengths have been weakened or
disappeared.
4. Conclusion
Bioremediation is a feasible method for the remediation of sand
containing soils highly contaminated with gasoil. More than 70% of
TPH could be removed from sandy soil in 270 days. The initial TPH
content was 7%. The bioremediation of clay soil was not successful
due to the low bioavailability of hydrocarbons to microorganisms.
Bioremediation of soils containing both clay and sand gave com-
parable results to the bioremediation of sandy soils. The moisture
content of 10% was sufficient for bioremediation and greater
moisture contents did not result in better performance. For quan-
tification of the removal percentage of TPH in soil the EPA Method
9071B which is a gravimetric method, and comparison of GC
chromatograms of the extracted TPH before and after the process
gave similar results. Our future research in this area is focused on
the ways for better bioremediation of clay soils. Application of
surfactants is a possible way to desorb hydrocarbons from clay and
improve their bioavailability. Inclusion of cheap inducers such as
waste organic materials is another possible method to improve
bioremediation of clay soils. Microbes can proliferate using the
inducers and consume the hydrocarbons upon the exhaustion of
the inducers with a large population. Other research needs in this
area are quantification of oxygen transport in clay soils, and the
effect of oxidation reduction potential on bioremediation of
201
contaminated clay soils.
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