Sustainable Environment Research 27 (2017) 195e202

Contents lists available at ScienceDirect

Sustainable Environment Research

journal homepage: www.journals.elsevier.com/sustainable-
environment-research/

Original Research Article

Bioremediation capability and characterization of bacteria isolated

from petroleum contaminated soils in Iran
Golafarin Ghoreishi a, Abbas Alemzadeh b, *, Mohammad Mojarrad a,
Mohammad Djavaheri c
a
Biotechnology Centre, School of Agriculture, Shiraz University, Shiraz 7144165186, Iran
b
Department of Crop Production and Plant Breeding, School of Agriculture, Shiraz University, Shiraz 7144165186, Iran
c
Department of Plant Protection, School of Agriculture, Shiraz University, Shiraz 7144165186, Iran

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

Article history: This study was carried out to isolate bacteria for bioremediation of petroleum polluted soils. Five samples
Received 21 May 2016 were used for isolation in this study. They were four soil samples in addition to one kerosene sample. The
Received in revised form soil samples including soils contaminated by crude oil and gas oil and two soil samples with no outward
23 December 2016
contamination which were collected from Shiraz Oil Refinery sites. Seven strains were selected among
Accepted 6 March 2017
Available online 6 May 2017
the isolated colonies for further experiments. The selected isolates were cultured in standard succinate
medium (SSM) minimal medium in which 2.5% v/v kerosene was used as carbon source. In another
bacterial SSM culture, carbon, sulfur or nitrogen source was removed and 20% v/v kerosene added to
Keywords:
Bioremediation
check the ability of isolates to utilizekerosene as sole source for C, N and S. Finally, cultures of four strains
Kerosene with higher growth in modified SSM cultures were selected for GC analysis. In this study they were
Citrobacter sedlakii named C2 and C4 which were isolated from crude oil contaminated soil and SI1 and SI2 isolated from
Entrobacter hormeachei soils with no outward contamination. GC analysis showed that C2 could degrade 69% of 5% v/v kerosene
Entrobacter cloacae in 7 d, while C4 and SI1 degraded 48% and 42% of 5% v/v kerosene during this 7-d period respectively, and
the degradation ability of SI2 was 38% after 7 d. Analysis of 16S rRNA gene showed that C2 was close to
Citrobacter sedlakii, C4 and SI1 were related to Entrobacter hormeachei and SI2 was close to Entrobacter
cloacae, respectively.
© 2017 Chinese Institute of Environmental Engineering, Taiwan. Production and hosting by Elsevier B.V.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).

1. Introduction
The release of petroleum components to the environment is
considered as a pervasive issue and usually occurs as a result of
petroleum exploration, accidents, transportation, and leakage from
waste disposal or storage sites, or from industrial facilities [1]. This
is a severe problem in oil producing countries such as Iran. Ac-
cording to a report, about 1,500,000 m3 of soil has been contami-
nated by crude oil near Tehran Oil Refinery [2]. Since petroleum
contains hazardous chemicals such as benzene, toluene, ethyl
benzene, xylene, and naphthalene, it can be harmful to plants,
animals, and humans’ health [3]. Oil spills have destructive impact
* Corresponding author.
E-mail address: alemzadeh@shirazu.ac.ir (A. Alemzadeh).
Peer review under responsibility of Chinese Institute of Environmental
Engineering.
on soil ecosystems not only because of their toxicity but also
because hydrocarbons in the soil reduce oxygen tension and
consequently increases anaerobiosis which is harmful to plant
roots [4,5]. Petroleum contamination is treated by physical,
chemical and biological approaches. The first two methods have
limitations such as high costs, inefficacy and altering natural
ecosystem. Today, biological treatment is a more interesting pro-
cess to remove petroleum contamination [3]. Bioremediation is a
technique in which biological systems such as microorganisms are
applied to destroy or transform (degrade) harmful chemicals [6]. In
recent years, employing hydrocarbon degrading bacteria to clean a
petroleum contaminated soil has become a prevalent, efficient and
economical technique that converts toxic wastes to non-toxic end
products. This technique has reduced the harmful effects on health
and ecology. Additionally, it provides the ability to perform in situ
treatment without unduly disturbing native ecosystems, compared
to physical and chemical remediation methods [3,7]. Either

http://dx.doi.org/10.1016/j.serj.2017.05.002
2468-2039/© 2017 Chinese Institute of Environmental Engineering, Taiwan. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
196 G. Ghoreishi et al. / Sustainable Environment Research 27 (2017) 195e202
indigenous microorganisms of contaminated sites or new micro-
organisms can be applied in bioremediation [6].
Due to the hydrophobic property of a petroleum compound,
most microorganisms cannot utilize hydrocarbons and thus, cannot
be employed in bioremediation [8]. Characterization of the bacte-
rial strains living in petroleum contaminated sites can help
improving bioremediation efficiency [9]. Studies showed that mi-
croorganisms such as Pseudomonas spp., Bacillus spp., Micrococcus
spp., Rhodoccucos spp. Entrobacter spp. Mycobactena spp., Mod-
ococci spp. and Acinetobacter spp. can degrade petroleum com-
pounds [7,9e11]. However, such microorganisms degrade these
compounds in different rates. Therefore, it is necessary to evaluate
their biodegradation capability. In order to select more efficient
bacteria for bioremediation, several methods have been found to
evaluate biodegradation capability of hydrocarbon degrading bac-
teria, among which gas chromatography (GC) is more common
[5,10,12e14].
In this paper, we aimed to isolate and characterize bacteria from
petroleum contaminated soils in Iran and to investigate their ability
to bioremediate kerosene using GC. Finally, we planned to check
the growth ability of isolates in high concentration of kerosene
(20% v/v) and utilizing it as carbon, sulfur and nitrogen sources.
2. Materials and methods
2.1. Sampling, culture media, and growth conditions
Four soil samples were used in this study which were taken
from the surface layer of soil with crude oil and gas oil contami-
nation, and two different sites without contamination. In addition,
kerosene was also used as the energy source. All samples were
collected from Shiraz Oil Refinery site (Shiraz, Iran, 29
440 200 N
52
390 2000 E). Shiraz is one of the big cities in Iran and has mild
weather. According to Islamic Republic of Iran Meteorological Or-
ganization, the daily average temperature of Shiraz is 18.0
C [15].
The heavy crude oil extracted from fields in southern Iran con-
tains high concentrations of sulfur and nitrogen compounds. Ac-
cording to the reports of Petroleum Ministry of Islamic Republic of
Iran, the kerosene produced in Iranian refineries contains a
considerable amount of sulfur compounds [16]; thus, we tested
sulfur biodegradability of our isolates using kerosene. Furthermore,
with the assumption of some nitrogen impurities, we also check the
ability of our isolates to biodegrade the nitrogen component pre-
sent in the kerosene.
2xYT [17] and SSM (standard succinate medium (in g L1): 6
K2HPO4, 3 KH2PO4, 4 succinic acid, 1 (NH4)2SO4 and 0.2 MgSO4, pH
7) [18] are the culture media used in this test. All culture media
were autoclaved at 120
C for 20 min. The liquid cultures were
incubated in a rotary shaker with 170 rpm. The temperature of
incubation for both liquid and solid cultures was 28
C.
2.2. Isolation
In order to isolate bacteria from kerosene, a 2xYT liquid medium
was prepared and kerosene was added in five concentrations of 10,
20, 30, 40 and 50% (v/v). After 24 h, 50 mL of the cultures were
inoculated to 1.5% agar 2xYT plates and incubated for 24 h to
observe colonies. Bacterial isolation from soil was carried out by
suspending 0.5 g of each soil sample in 5 mL 2xYT. After 24 h, 1 mL
of the culture was serially diluted and plated on 1.5% agar 2xYT
plates, and the colonies were checked after 24-h incubation at
28
C.
Sixty-seven and eighty-five colonies were isolated from kero-
sene and petroleum-contaminated soils, respectively. Seven col-
onies with different morphological features were selected from
kerosene and contaminated soils. The selected isolates were named
as K1, C2, C4, G2, SI1, SI2 and SII2; the originated source of these 7
isolates is listed in Table 1.
2.3. Growth test using 2.5% v/v kerosene
Each selected isolate was inoculated into a 5 mL liquid SSM and
incubated overnight. Then, the cultures were centrifuged at
5000 rpm for 5 min in the room temperature. The obtained pellet of
each isolate was resuspended in a modified SSM having 2.5% v/v
kerosene as the sole source of carbon (called as modified SSM-2.5C
medium, see Table 2), until the optical density of the cultures at
600 nm reached almost 0.5, equaling to about 5  108 colony
forming unit (CFU) mL1. The growth of each isolate was monitored
every 24 h by UV/Vis spectrophotometer SP-3000 Plus (Optima,
Tokyo, Japan) in 3 d.
2.4. Growth test using 20% v/v kerosene
To further check the growth ability of microbes in highly
kerosene-contaminated media, three considerable tests of
removing carbon, sulfur and nitrogen sources were conducted. For
the test of carbon removal, the addition of 20% v/v kerosene was
used to replace succinic acid in SSM medium (named as modified
SSM-20C medium). For the test of sulfur removal, (NH4)2PO4, MgCl2
and 20% v/v kerosene were added, while (NH4)2SO4 and MgSO4
were removed from SSM (called as modified SSM-20S medium).
Finally, the test of nitrogen removal was conducted using an SSM
medium with 20% v/v of kerosene and Na2SO4, instead of (NH4)2SO4
in SSM medium (named as modified SSM-20N medium). A fresh
liquid culture of each selected isolate (3000e6000 CFU mL1) was
inoculated into 1 mL of modified SSM media, SSM-20C, SSM-20S
and SSM-20N. Two additional cultures were set up for isolates in
each test; positive and negative control cultures. The positive
control culture was the unmodified SSM, while the negative control
cultures were prepared by removing carbon, sulfur, or nitrogen
source from SSM medium, respectively to the corresponding test,
e.g., succinic acid was removed for the test of carbon removal (see
Table 2). The positive and negative control media were both kero-
sene free. The description of different modified SSM media used in
this work are presented in Table 2. After 48 h of the experimental
period, the growth of isolates in three tests was measured by
UVevis spectrophotometer SP-3000 Plus at 600 nm.
2.5. GC analysis
The sample used for GC analysis was prepared as follow. A
250 mL of fresh bacterial culture was inoculated into a 25 mL SSM
and incubated for 24 h. And then, the mixture was centrifuged at
5000 rpm for 5 min in the room temperature. For the test of carbon
removal, the pellet was resuspended in 25 mL SSM containing 5% v/
Table 1
Selected isolates for bioremediation tests.
Isolate Originated source
K1 Kerosene
C2 Crude oil
C4 Crude oil
G2 Gas oil
SI1 Polluted soil with no outward
contamination from location I
SI2 Polluted soil with no outward
contamination from location I
SII2 Polluted soil with no outward
contamination from location II
 G. Ghoreishi et al. / Sustainable Environment Research 27 (2017) 195e202 197

Table 2 Table 3
The description of SSM media used in the different growing tests. OD600 of isolates in SSM with 2.5% v/v kerosene as carbon source.

Medium Description Isolate Day 1 Day 2 Day 3

Modified SSM-2.5C SSM in which succinic acid was removed and K1 0.369 0.688 0.641
2.5% v/v kerosene was added. C2 0.712 1.276 1.468
Modified SSM-20C SSM in which succinic acid was removed and C4 0.692 0.754 0.856
20% v/v kerosene was added. SI1 0.602 0.879 0.902
Modified SSM-5C SSM in which succinic acid was removed and 5% SI2 0.485 0.609 0.619
v/v kerosene was added. G2 0.779 0.782 0.807
Modified SSM-0C SSM in which succinic acid was removed SII2 0.491 0.602 0.699
Modified SSM-20S SSM in which (NH4)2SO4 and MgSO4 were
OD600 of all isolates was 0.5 in day 0.
removed and (NH4)2PO4a, MgCl2a and 20% v/v
kerosene were added
Modified SSM-5S SSM in which (NH4)2SO4 and MgSO4 were
removed and (NH4)2PO4a, MgCl2a and 5% v/v
kerosene were added
Modified SSM-0S SSM in which (NH4)2SO4 and MgSO4 were
removed
Modified SSM-20N SSM in which (NH4)2SO4 was removed and
Na2SO4a and 20% v/v kerosene were added
Modified SSM-0N SSM in which (NH4)2SO4 was removed
SSM Unmodified SSM
a
The compounds added in order to minimize the alteration of SSM composition.

v kerosene instead of succinic acid (called as modified SSM-5C

medium). For the test of sulfur removal, the respective pellet was
resuspended in a modified SSM having 5% v/v kerosene instead of
(NH4)2SO4 and MgSO4 (named as modified SSM-5S medium). The
resuspended cultures were incubated at 28
C and shaking at
170 rpm for 7 d. It should be noted that such a GC test was not
reported for the nitrogen removal test, since the results for this test
were not satisfactory.
After 7 d, kerosene was extracted with equal volume of n-hex-
ane for all samples. Then, 0.1 mL of each extract was injected into a
GC (model 3700 of Varian, USA) equipped with a 10% SP 2100
capillary column (Supelco, USA). In this work, the GC program was
used according to the report published by Wongsa et al. [19]. The
Fig. 1. OD600 of the isolates in the modified SSM-2.5C medium.
area of each peak in the chromatogram was obtained and analyzed
by Chem Station A.08.03 [847] software. The percentage of
biodegradation was calculated by the following relation based on Partial sequences of 16S rRNA genes were amplified by PCR
total area of each sample per control analysed using the identical (Polymerase chain reaction) using fDI upstream primer (30 -
conditions [18]: AGAGTTTGATCCTGGCTCAG-50 ) and rP2 downstream primer (30 -
ACGGCTACCTTGTTACGACT-50 ) [20]. PCR was performed in 30 mL
Sum of the peak areas of tested sample
 100 volumes using 2.5 mL of 10 reaction buffer (10 mM TriseHCl (pH
Sum of the peak areas of control sample 8.3), 50 mM KCl), 1.5 mM MgCl2, 200 mM dNTP, 0.3 mM of each
primer, 0.5 U Taq DNA polymerase (CinnaGen, Tehran, Iran).
Amplification was carried out in a Bioer Gene Pro PCR thermocycler
2.6. Biochemical characterization (Bioer Technology, Binjiang, P.R. China) using the following pro-
gram: (1) 5 min at 94
C for initial denaturation, (2) a run of 30
The biochemical tests used in this study to characterize the cycles, with each cycle consisting of 30 s denaturation at 94
C, 30 s
bacteria, including Gram reaction, Oxidative Fermentative test, annealing at 62
C, 2 min extension at 72
C, and (3) 10 min at 72
C
production of florescent pigment, motility, catalase, and oxidase. To for final extension. Total volume of reactions was 30 mL. After
accurate the biochemical characterization, API20E kit (bioMe'rieux, electrophoresis, PCR products in the gel were extracted by GF-1
Marcy l’Etoile, France) was applied and the obtained results were Nucleic Acid Extraction Kit (Vivantis, Selangor DarulEhsan,
analysed by apiweb™ software.

Table 4
2.7. 16S rRNA gene analysis
OD600 of isolates in SSM with 20% kerosene as carbon source.

Bacterial DNAs were extracted using modified cetyl- Isolate Modified SSM-20C SSM Modified SSM-0C

trimethylammonium bromide method. 6 mL of lysozyme K1 0.362 b

0.534 b
0.002c
(30 mg mL1) was used to break the cell wall of bacteria and 15 mL C2 0.253b 0.487a 0.005c
0.878a 0.487a 0.001b
of 20% sodium dodecyl sulphate and 3 mL proteinase K C4
SI1 0.702a 0.585a 0.006b
(20 mg mL1) were added to the mixture. Then, 100 mL NaCl (5 M) SI2 0.506a 0.361a 0.002b
and 80 mL of extraction buffer were used. After incubation in water G2 0.186a 0.206a 0.001b
bath at 65
C for 10e15 min, the treatment of the extracted mixture SII2 0.402a 0.373a 0.002b
was followed by addition of 0.5 mL chloroform: isoamyl alcohol Same lowercase letters are not statistically different among culture media (in a row)
(24:1) and 0.7 mL of phenol: chloroform: isoamyl alcohol (25:24:1). at p < 0.05.
198 G. Ghoreishi et al. / Sustainable Environment Research 27 (2017) 195e202

Table 5 3. Results and discussion

OD600 of isolates in SSM with 20% kerosene as sulfur source.

Isolate Modified SSM-20S SSM Modified SSM-0S 3.1. Isolation and contamination sources
K1 0.750a 0.558b 0.598b
C2 0.289ab 0.035b 0.581a As previously mentioned, some specific types of bacteria being
C4 0.841a 0.538a 0.571a able to degrade compounds usually are found in the petroleum
SI1 0.746a 0.476b 0.527b contaminations, and they choose the compounds to utilize for their
SI2 1.745a 0.254b 0.471b
growth [11]. The compounds such as crude oil, gas oil, polyaromatic
G2 0.551a 0.441b 0.469b
SII2 0.473a 0.440a 0.370a hydrocarbons (PAHs), benzene, toluene and naphthalene as energy
source have been reported by some research groups [7,9,18,21,22];
Same lowercase letters are not statistically different among culture media (in a row)
at p < 0.05.
additionally, kerosene has also been discussed as a useful energy
source by others [19,23,24]. In this study, we isolated microbes
using kerosene because it is not only widely used as a petroleum
Malaysia). The purified PCR products were sequenced at Bio Basic,
product, but also contained lot of environmentally harmful com-
Canada, and then obtained sequences were analysed by Vector NTI
pounds, e.g., n-alkanes, cycloalkanes, PAHs, benzene, toluene and
suite 9 (Life Technologies Corporation, invitrogen™, Grand Island,
xylene [25,26].
USA) software and BLASTN 2.2.26 (Basic Local Alignment Search
A variety of microbes having the degrading capacity were iso-
Tool) program in National Centre for Biotechnology Information
lated from petroleum contaminated soils (especially crude oil
website (http://blast.ncbi.nlm.nih.gov).
contaminated soil) [27]. This variety helps in choosing a diverse
consortium of microorganisms which results in a more efficient
2.8. Statistical analysis bioremediation process. In this work, a variety of soils with
different contamination sources was used for bacterial isolation to
The obtained data were analysed by SAS 9.1. The design of ex- obtain a diverse microbial consortium, since a single strain cannot
periments was completely randomized with five replications. The degrade all the components of a petroleum contamination source
means were compared by Duncan test at confidence level of 95%. [9]. In this regard, as mentioned in the following sections, the

Fig. 2. The component variation of crude kerosene by gas chromatogram before and after the degradation of C. sedlakii cultured in the modified SSM-5C medium at 28
C for 7 d.
 G. Ghoreishi et al. / Sustainable Environment Research 27 (2017) 195e202 199

Table 6 other isolates increased. An increase in the growth of K1, SI2 and
OD600 of isolates in 20% kerosene SSM as nitrogen source. SII2 was observed in the 2nd day, while all other isolates continued
Isolate Modified SSM-20N SSM Modified SSM-0N to grow. The growth of C2 increased in the 3rd day, whereas that of
K1 0.113b 0.588a 0.002c
others stopped or decreased. The most remarkable growth was
C2 0.053b 0.477a 0.002b observed for C2 and SI1 in 2.5% v/v kerosene, while K1 and SI2
C4 0.030b 0.627a 0.000b showed lower growths.
SI1 0.038b 0.723a 0.001b Table 4 presented that 20% v/v of kerosene was added instead of
SI2 0.036b 0.636a 0.010b
the carbon source in SSM medium (called as modified SSM-20C
G2 0.025b 0.333a 0.011b
SII2 0.016b 0.360a 0.001b medium) in our tests. Results showed that all cultured samples
had a significant growth in modified SSM-20C medium. The high
Same lowercase letters are not statistically different among culture media (in a row)
at p < 0.05.
growth for C4, SI1, SI2 and SII2 was observed, wherein C4 was up to
0.878; while, the growth for K1, C2 and G2 was lower than that
cultured in SSM medium. However, the lower growth of K1, C2 and
isolates which were taken from crude oil contaminated soil (C2 and G2 in modified SSM-20C medium was not statically significant in
C4) represent a higher biodegradation capability. That was because comparison with that of in SSM medium as noted in Table 4.
many microbial species survived in a crude oil contaminated soil, Obviously, no isolate could grow in SSM without kerosene and
which lead to a more efficient petroleum biodegradation. carbon source (succinic acid).
Our isolates grew in 2.5% (v/v) and 20% (v/v) of kerosene, higher
3.2. Growing tests using kerosene than the concentration (0.1%) used in the studies of others [23,24],
and that (1%) reported by Wongsa et al. [19]. In the tests of carbon
The growth of isolates in modified SSM-2.5C medium is re- removal, all strains could grow in modified SSM-2.5C medium and
ported in Table 3 and depicted in Fig. 1. A reduction in the growth of modified SSM-20C medium. Interestingly, C4, SI1, SI2 and SII2 grew
K1 was observed in the 1st day whereas, no significant change in more vigorously in modified SSM-20C medium than in SSM;
the OD600 of SI2 and SII2 was observed. Meanwhile, the growth of however, it was not statistically significant as noted in Table 4. High

Fig. 3. The profile for the degradation of crude kerosene by gas chromatogram after E. hormachei (SI1 and C4) cultured in the modified SSM-5C medium at 28
C for 7 d.
200 G. Ghoreishi et al. / Sustainable Environment Research 27 (2017) 195e202

Fig. 4. The component variation of crude kerosene by gas chromatogram before and after the degradation of E. cloacae cultured in the modified SSM-5S medium at 28
C for 7 d.

OD value of 0.8e1.0 for bacterial growth had been reported in 1%
crude oil [28]; however, to the best of our knowledge, the bacterial
growth in SSM medium containing petroleum compounds was not
yet compared to that in SSM medium.
The OD values of isolates in modified SSM-20S medium are
summarized in Table 5. The growth for all isolates in modified SSM-
20S medium was higher than that cultured in SSM, wherein the
highest growth was detected in the isolate SI2. Other isolates
except for C4 and SII2 could more efficiently grow in modified SSM-
20S medium than SSM. The growth of all isolates except C2 in
modified SSM-0S medium (see Table 2) was poor than that in the
modified SSM-20S medium; whereas, C2 grew considerably in
modified SSM-0S medium.
From the above results, we summarize that the isolates cultured
in this study could utilize kerosene as sulfur source. This result was
in agreement with the data reported by others [29e32], in which
sulfur source was originated from crude oil or contaminants.
Nevertheless, we did not extract sulfur compounds from kerosene
to be used directly in the tests, but the sulfur source was included in
the kerosene. In this way, the original conditions of a petroleum
contaminated site could be simulated for our strains.
The results for the tests of nitrogen removal in modified SSM-
20N medium are shown in Table 6. K1 was the only one to grow
in modified SSM-20N medium; however, its growth was signifi-
cantly lower in comparison to SSM medium. On the contrary, some
research groups pointed out that microbes could effectively utilize
petroleum as nitrogen sources [29,31]. Furthermore, the growth of
K1 for nitrogen degradation was much lower than that those for
carbon and sulfur removal. This is probably due to the three
following reasons. First, nitrogen compounds were not extracted
from kerosene to be directly applied in this test. Second, nitrogen is
one of the essential minerals for bacterial growth and helps to

Table 7
The results of biochemical and API 20E kit.

Isolate Gram OF Oxidase Catalase Motility Production of API 20E kit

fluorescent pigment

C2 e Facultative e þ þ e Citrobater koseri/farmeri

C4 e Facultative e þ þ e Entrobacter cloacae
SI1 e Facultative e þ þ e Entrobacter cloacae
SI2 e Facultative e þ e e Enterobacter cloacae
 G. Ghoreishi et al. / Sustainable Environment Research 27 (2017) 195e202
improve bioremediation process through stimulating a bacterial
community to degrade petroleum pollutants [33,34]. We had
assumed the kerosene used in our study to contain small amounts
of nitrogen, so that, the nitrogen source was completely removed
from SSM and kerosene was added. Nevertheless, the isolates could
not grow in modified SSM-20N medium. Therefore, the concen-
tration of nitrogen in the kerosene might be less than that needed
for bacterial growth. In fact, microorganisms may require some
primary nitrogen source to grow. Third, compared to other works,
20% v/v kerosene was probably high, so that, the capacity of ni-
trogen utilization for isolates was declined [10,19,23,24]. The
mechanism of nitrogen utilization in the petroleum compounds for
microbes will be further investigated in the future.
3.3. GC analysis
For GC analyses, we selected isolates C2, C4 and SI1 due to their
high growth in the growing tests with 2.5% v/v of kerosene; in
addition, SI2 was considered to be one of the selections because it
vigorously grew in modified SSM-20S medium. The results revealed
that all isolates could utilize 5% v/v of kerosene as carbon source,
and kerosene could be a sulfur source for SI2. The degradation ef-
ficiencies for C2, C4 and SI1 in 7 days were 69, 47 and 42%,
respectively (Figs. 2 and 3). In addition, the removal rate of sulfur
compound for SI2 was 38% after 7 d (Fig. 4). Similar degradation
efficiency was observed in C4 and SI1, probably due to their close
phylogenetic relationship. For the kerosene degradation, 95%
removal of 2% v/v kerosene for microbes in 21 d was reported by
Gouda et al. [24], and Wangsa et al. [19] indicated 50% degradation
efficiency of 1% v/v kerosene in one week. Based on the above
analysis, we concluded that our isolates had a high potential for the
rapid degradation of high kerosene concentration.
3.4. Characterization of isolates
Results of biochemical characterization using API 20E kit are
summarized in Table 7. 16S rRNA analysis showed that C2 was
related to Citrobacter sedlakii (Accession No. GU726186.1, 97%
similarity), and both of C4 and SI1 were close to Entrobacter hor-
meachei (Accession No. EU239467.1, 99% similarity). On the other
hand, SI2 closely belonged to Entrobacter cloacae (Accession No.
KC764978.1, 99% similarity).
The results obtained using API 20E kit showed that C2, C4 and
SI1 were related to Citrobater koseri/farmer, E. cloacae and E. cloacae,
respectively. The API 20E kit is a biochemical method for bacterial
characterisation. However, these results are not consistent with 16S
rRNA gene analysis which is a genetic characterisation method and
seems to be more accurate [35]. Therefore, here, we have consid-
ered the results of the 16S rRNA analysis. Furthermore,
E. hormeachei had very closely phylogenetic relationship with
E. cloacae based on the analysis of 16S rRNA sequences.
Results of characterization tests indicated that C2, C4, and SI2
were identified as C. sedlakii, E. hormeachei and E. cloacae, respec-
tively. Also, the characterization of SI1 was similar to E. hormeachei.
Citrobacter spp. and Entrobacter spp. that could degrade petroleum
compounds [10,36e38], and the degrading ability for E. hormeachei
had rarely been discussed [39]; however, the degrading pathway for
C. sedlakii was unclear. Additionally, E. cloacae had the ability for the
degradation of petroleum compounds like alkanes and PAHs [10,40].
4. Conclusions
This study investigated the degradation potential of the bacteria
isolated from kerosene and petroleum-contaminated soils for
kerosene treatment. It was revealed that these isolated bacteria
201
were able to grow highly in modified SSM media with 20% v/v of
kerosene (modified SSM-20C and SSM-20S). According to the GC
results, these isolates could also considerably degrade carbon and
sulfur compounds of 5% v/v kerosene in 7 d. Results of GC analysis
showed that C2 isolated from crude-oil contaminated soil, which
was related to C. sedlakii (97% similarity), had the potential for
carbon degradation in kerosene. In addition, SI2 isolated from soil
with no outward contamination presented the high potential for
sulfur degradation in kerosene, and it was closely related to E.
cloacae (99% similarity). Using these two strains in a microbial
consortium may be helpful in conducting a more efficient biodeg-
radation process, since together, they are able to degrade two main
polluting components found in a petroleum contaminated soil.
Our study opens a good viewpoint for more advanced and
specified research in the context of petroleum biodegradation. In
this regard, conducting in situ tests can improve the results of the
present work. Additionally, isolating microbes from other
petroleum-contaminated sites with different climates is suggested
in order to find the most efficient microbial consortium for
biodegradation of petroleum pollution.
Acknowledgement
The authors are thankful to Shiraz University for supporting this
research.
References
[1] Khan FI, Husain T, Hejazi R. An overview and analysis of site remediation
technologies. J Environ Manage 2004;71:95e122.
[2] Kebria DY, Khodadadi A, Ganjidoust H, Badkoubi A, Amoozegar MA. Isolation
and characterization of a novel native Bacillus strain capable of degrading
diesel fuel. Int J Environ Sci Technol 2009;6:435e42.
[3] Sarkar D, Ferguson M, Datta R, Birnbaum S. Bioremediation of petroleum
hydrocarbons in contaminated soils: comparison of biosolids addition, carbon
supplementation, and monitored natural attenuation. Environ Pollut
2005;136:187e95.
[4] Bossert I, Bartha R. The fate of petroleum in the soil ecosystem. In: Atlas RM,
editor. Petroleum Microbiology. New York: Macmillan; 1984. p. 435e75.
[5] Ekpo MA, Udofia US. Rate of biodegradation of crude oil by microorganisms
isolated from oil sludge environment. Afr J Biotechnol 2008;7:4495e9.
[6] Crawford RL. Bioremediation. In: Dworkin M, editor. The Prokaryotes, Volume
1: Symbiotic Association, Biotechnology, Applied Microbiology. New York:
Springer; 2006. p. 48e93.
[7] Mirdamadian SM, Emtiazi G, Golabi MH, Ghanavati H. Biodegradation of pe-
troleum and aromatic hydrocarbons by bacteria isolated from petroleum-
contaminated soil. J Petrol Environ Biotechnol 2010;1:1e5.
[8] Lin TC, Shen FT, Chang JS, Young CC, Arun AB, Lin SY, et al. Hydrocarbon
degrading potential of bacteria isolated from oil-contaminated soil. J Taiwan
Inst Chem Eng 2009;40:580e2.
[9] Zhang ZZ, Gai LX, Hou ZW, Yang CY, Ma CQ, Wang ZG, et al. Characterization
and biotechnological potential of petroleum-degrading bacteria isolated from
oil-contaminated soils. Bioresour Technol 2010;101:8452e6.
[10] Saadoun I. Isolation and characterization of bacteria from crude petroleum oil
contaminated soil and their potential to degrade diesel fuel. J Basic Microb
2002;42:420e8.
[11] Thapa B, Kumar KCA, Ghirmire A. A review on bioremediation of petroleum
hydrocarbon contaminants in soil. Kathmandu Univ J Sci Eng Technol 2012;8:
164e70.
[12] Zawierucha I, Malina G. Effects of oxygen supply on the biodegradation rate in
oil hydrocarbons contaminated soil. J Phys Conf Ser 2011;289:1e9.
[13] Owsianiak M, Chrzanowski L, Szulc A, Staniewski J, Olszanowski A, Olejnik-
Schmidt AK, et al. Biodegradation of diesel/biodiesel blends by a consortium
of hydrocarbon degraders: effect of the type of blend and the addition of
biosurfactants. Bioresour Technol 2009;100:1497e500.
[14] Nikolopoulou M, Pasadakis N, Kalogerakis N. Enhanced bioremediation of
crude oil utilizing lipophilic fertilizers. Desalination 2007;211:286e95.
[15] IRIMO. 2014 Analytical Reports and Climatic Data. Tehran, Iran: Islamic Re-
public of Iran Meteorological Organization; 2016 [in Persian].
[16] MOP. Crude Oil, Petroleum Products & Petrochemical Trading Guide Book.
Tehran, Iran: Ministry of Petroleum; 2013.
[17] Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual.
New York: Cold Spring Harbor Laboratory; 1989.
[18] Lee YH, Lee WH, Lee DK, Shim HK. Factors relating to induced systemic
resistance in watermelon by plant growth-promoting Pseudomonas spp. Plant
Pathol J 2001;17:174e9.
202 G. Ghoreishi et al. / Sustainable Environment Research 27 (2017) 195e202
[19] Wongsa P, Tanaka M, Ueno A, Hasanuzzaman M, Yumoto I, Okuyama H.
Isolation and characterization of novel strains of Pseudomonas aeruginosa and
Serratia marcescens possessing high efficiency to degrade gasoline, kerosene,
diesel oil, and lubricating oil. Curr Microbiol 2004;49:415e22.
[20] Weisburg WG, Barns SM, Pelletier DA, Lane DJ. 16s ribosomal DNA amplifi-
cation for phylogenetic study. J Bacteriol 1991;173:697e703.
[21] Ridgway HF, Safarik J, Phipps D, Carl P, Clark D. Identification and catabolic
activity of well-derived gasoline-degrading bacteria from a contaminated
aquifer. Appl Environ Microb 1990;56:3565e75.
[22] Parales RE, Ditty JL, Harwood CS. Toluene-degrading bacteria are chemotactic
towards the environmental pollutants benzene, toluene, and trichloroethy-
lene. Appl Environ Microb 2000;66:4098e104.
[23] Vennila R, Kannan V. Bioremediation of petroleum refinery effluent by Pla-
nococcus halophilus. Afr J Biotechnol 2011;10:8829e33.
[24] Gouda MK, Omar SH, Chekroud ZA, Hemdan M, Eldin N. Bioremediation of
kerosene I: a case study in liquid media. Chemosphere 2007;69:1807e14.
[25] Dror I, Gerstl Z, Yaron B. Temporal changes in kerosene content and compo-
sition in field soil as a result of leaching. J Contam Hydrol 2001;48:305e23.
[26] Di Martino C, Lopez NI, Iustman LJR. Isolation and characterization of benzene,
toluene and xylene degrading Pseudomonas sp selected as candidates for
bioremediation. Int Biodeter Biodegr 2012;67:15e20.
[27] Hamamura N, Olson SH, Ward DM, Inskeep WP. Microbial population dy-
namics associated with crude-oil biodegradation in diverse soils. Appl Environ
Microb 2006;72:6316e24.
[28] Rahman KSM, Thahira-Rahman J, Lakshmanaperumalsamy P, Banat IM. To-
wards efficient crude oil degradation by a mixed bacterial consortium. Bio-
resour Technol 2002;85:257e61.
[29] Das K, Mukherjee AK. Crude petroleum-oil biodegradation efficiency of Bacillus
subtilis and Pseudomonas aeruginosa strains isolated from a petroleum-oil
contaminated soil from North-East India. Bioresour Technol 2007;98:1339e45.
[30] Gallego JR, Gonzalez-Rojas E, Pelaez AI, Sanchez J, Garcia-Martinez MJ,
Ortiz JE, et al. Natural attenuation and bioremediation of Prestige fuel oil along
the Atlantic coast of Galicia (Spain). Org Geochem 2006;37:1869e84.
[31] Mishra S, Jyot J, Kuhad RC, Lal B. Evaluation of inoculum addition to stimulate
in situ bioremediation of oily-sludge-contaminated soil. Appl Environ Microb
2001;67:1675e81.
[32] Duarte GF, Rosado AS, Seldin L, de Araujo W, van Elsas JD. Analysis of bacterial
community structure in sulfurous-oil-containing soils and detection of species
carrying dibenzothiophene desulfurization (dsz) genes. Appl Environ Microb
2001;67:1052e62.
[33] Evans FF, Rosado AS, Sebastian GV, Casella R, Machado PLOA, Holmstrom C,
et al. Impact of oil contamination and biostimulation on the diversity of
indigenous bacterial communities in soil microcosms. FEMS Microbiol Ecol
2004;49:295e305.
[34] Boopathy R. Factors limiting bioremediation technologies. Bioresour Technol
2000;74:63e7.
[35] Bosshard PP, Zbinden R, Abels S, Boddinghaus B, Altwegg M, Bottger EC. 16S
rRNA gene sequencing versus the API 20 NE system and the VITEK 2 ID-GNB
card for identification of nonfermenting gram-negative bacteria in the clinical
laboratory. J Clin Microbiol 2006;44:1359e66.
[36] Dindar E, Şag ban FOT, Başkaya HS. Bioremediation of petroleum-
contaminated soil. J Biol Environ Sci 2013;7:39e47.
[37] Orji FA, Ibiene AA, Dike EN. Laboratory scale bioremediation of petroleum
hydrocarbon e polluted mangrove swamps in the Niger Delta using cow
dung. Malays J Microb 2012;8:219e28.
[38] Mojarrad M, Alemzadeh A, Ghoreishi G, Javaheri M. Kerosene biodegradation
ability and characterization of bacteria isolated from oil-polluted soil and
water. J Environ Chem Eng 2016;4:4323e9.
[39] Erdog an EE, Şahin F, Kacara F. Determination of petroleum-degrading bacteria
isolated from crude oil-contaminated soil in Turkey. Afr J Biotechnol 2012;11:
4853e9.
[40] Hua XF, Wu ZJ, Zhang HX, Lu DN, Wang M, Liu YM, et al. Degradation of
hexadecane by Enterobacter cloacae strain TU that secretes an exopoly-
saccharide as a bioemulsifier. Chemosphere 2010;80:951e6.