in sterile tubes containing ONR7a liquid medium
supplemented respectively with 1% (v/v) of crude oil, nhexadecane (C 16 ), n-hexacosane (C 26 ), n-octacosane
(C 28 ), benzene, toluene and xylene as sole carbon and
energy source. As a control hydrocarbon-free medium
was used and a control was set up for each trial. Growth
was evaluated after 1 week of incubation at 30 Æ 1°C by
measuring the turbidity (OD at 600 nm) using a spectrophotometer. The degradation ability of selected strains
was evaluated in culture medium ONR7a supplemented
with crude oil. Starter cultures were prepared by inoculating a single colony into ONR7a medium supplemented
with 1% (v/v) sterile hexadecane as the sole carbon
source. After 1 week of incubation at 30 Æ 1°C in a
rotary shaker, cells were centrifuged at 9000 g for
10 min. The pellet was then resuspended in sterile medium to an OD (600 nm) of 0Á1 and used as inoculum for
the biodegradation experiment. Using 100 ml flasks, 1 ml
of the suspension was inoculated into 30 ml of ONR7a
supplemented with 30 ll filter-sterilized crude oil. A flask
without any inoculum was used as an abiotic control. All
flasks were incubated in a rotary shaker at 30°C for
1 week. Residual hydrocarbons were extracted using a
continuous liquid–liquid extraction technique, and analysed by high-resolution GC–FID following the 3510 EPA
(Environmental Protection Agency). After acidification of
the samples, dichloromethane (CH 2 Cl 2 , Sigma-Aldrich,
Milan, Italy; 10% v/v) was added to the culture flask and
homogenized on a shaking table at room temperature.
The procedure was repeated three times, and the solvent
phase was combined and dehydrated with anhydrous
sodium sulphate (Na 2 SO 4 , Sigma-Aldrich). The extracts
were concentrated by rotary evaporation (Rotavapor
model R110; B€ uchi Labortechnik AG, Flawil, Switzerland)
at room temperature. All measurements were performed
using a DANI Master GC Fast Gas Chromatograph System (DANI Instruments S.p.A., Milan, Italy) equipped
with a split/splitless SSL injector and FID detector (EPA
1996; Gentile et al. 2016).
Detection of catabolic genes in selected bacteria
In order to detect the catabolic genes involved in
biodegradation pathways of hydrocarbons, PCR amplification of monooxygenase genes fragments was performed.
The fragment of the terminal hydroxylase component of
xylene monooxygenase encoded by the xylM gene, and
the electron transfer component of the xylene monooxygenase encoded by the xylA gene was amplified using primer sets TOL-F/TOL-R and XYLA-F/XYLA-R respectively
as described by (Hendrickx et al. 2006). A fragment of
the alkane monooxygenase gene alkB was amplified by
PCR. The amplification was carried out by using the
primers set alkB-1f and alkB-1r (Kloos et al. 2006) in a
total volume of 30 ll containing: 1 ll DNA, 0Á5 lmol
l
À1 of each forward and reverse primer, 0Á2 mmol l
À1
dNTPs, 19 phire reaction buffer and 0Á6 ll of Phire Hot
Start II DNA Polymerase (Thermo Scientific). The PCR
conditions used were as follows: an initial denaturation at
98°C for 30 s, followed by 35 cycles of (98°C for 15 s,
55°C for 15 s, 72°C for 10 s) and a final extension at
72°C for 1 min. All amplicons were visualized on 1%
agarose gel.
Biosurfactant production and emulsification test
Two distinct methods were used for the screening of the
biosurfactant production by isolates: (i) the drop collapse
test and (ii) the oil spreading method. The drop collapse
test was performed by adding 7 ll of crude oil into each
well of a 96-well microtitre plate lid. The lid was equilibrated for 1 h at room temperature, and then 20 ll of
the culture was added to the surface of oil. After 1 min
of incubation, the shape of the drop on the surface of the
oil was observed. If the drop collapses, it indicates the
presence of surfactant (positive result), if it remains
beaded, it indicates the absence of surfactant (negative
response). Sodium dodecyl sulphate with the sterile distilled water was used as positive and negative control
respectively (Bodour and Miller-Maier 1998; Mahjoubi
et al. 2013). In the oil spreading method, 20 ml of distilled water was added to an empty Petri dish (90 mm),
and 10 ll of crude oil was added to the surface of water
to form a thin layer of hydrocarbons on the surface. The
following step was to apply 10 ll of bacterial culture on
the oil surface. The diameter of clear zone around the
bacterial suspension was measured as the activity of surfactants (Cappello et al. 2016). The emulsification index
(E24) of different bacterial culture was determined by
adding 2 ml of hexadecane to the same amount of bacterial culture. The mixture was vigorously mixed (vortex)
for 2 min. The mixture could stand for 24 h prior to
measurement. The emulsification activity was calculated
as a percentage of the height of the emulsified layer
divided by the total height of the liquid column (Hassanshahian et al. 2012).
Results
Toxicity assay
The bioluminescence inhibition assay (Microtox) based
on the Gram-negative bacterium V. fisheri was used to
establish the toxicity level of the polluted marine sediment. The data revealed that all samples inhibited the
bioluminescence of V. fisheri more than 20% and their
Journal of Applied Microbiology 126, 780--795 © 2018 The Society for Applied Microbiology
784
The use of oil-degrading bacteria as an alternative for environmental remediation (bioremediation)
N. Djahnit et al.
supplemented respectively with 1% (v/v) of crude oil, nhexadecane (C 16 ), n-hexacosane (C 26 ), n-octacosane
(C 28 ), benzene, toluene and xylene as sole carbon and
energy source. As a control hydrocarbon-free medium
was used and a control was set up for each trial. Growth
was evaluated after 1 week of incubation at 30 Æ 1°C by
measuring the turbidity (OD at 600 nm) using a spectrophotometer. The degradation ability of selected strains
was evaluated in culture medium ONR7a supplemented
with crude oil. Starter cultures were prepared by inoculating a single colony into ONR7a medium supplemented
with 1% (v/v) sterile hexadecane as the sole carbon
source. After 1 week of incubation at 30 Æ 1°C in a
rotary shaker, cells were centrifuged at 9000 g for
10 min. The pellet was then resuspended in sterile medium to an OD (600 nm) of 0Á1 and used as inoculum for
the biodegradation experiment. Using 100 ml flasks, 1 ml
of the suspension was inoculated into 30 ml of ONR7a
supplemented with 30 ll filter-sterilized crude oil. A flask
without any inoculum was used as an abiotic control. All
flasks were incubated in a rotary shaker at 30°C for
1 week. Residual hydrocarbons were extracted using a
continuous liquid–liquid extraction technique, and analysed by high-resolution GC–FID following the 3510 EPA
(Environmental Protection Agency). After acidification of
the samples, dichloromethane (CH 2 Cl 2 , Sigma-Aldrich,
Milan, Italy; 10% v/v) was added to the culture flask and
homogenized on a shaking table at room temperature.
The procedure was repeated three times, and the solvent
phase was combined and dehydrated with anhydrous
sodium sulphate (Na 2 SO 4 , Sigma-Aldrich). The extracts
were concentrated by rotary evaporation (Rotavapor
model R110; B€ uchi Labortechnik AG, Flawil, Switzerland)
at room temperature. All measurements were performed
using a DANI Master GC Fast Gas Chromatograph System (DANI Instruments S.p.A., Milan, Italy) equipped
with a split/splitless SSL injector and FID detector (EPA
1996; Gentile et al. 2016).
Detection of catabolic genes in selected bacteria
In order to detect the catabolic genes involved in
biodegradation pathways of hydrocarbons, PCR amplification of monooxygenase genes fragments was performed.
The fragment of the terminal hydroxylase component of
xylene monooxygenase encoded by the xylM gene, and
the electron transfer component of the xylene monooxygenase encoded by the xylA gene was amplified using primer sets TOL-F/TOL-R and XYLA-F/XYLA-R respectively
as described by (Hendrickx et al. 2006). A fragment of
the alkane monooxygenase gene alkB was amplified by
PCR. The amplification was carried out by using the
primers set alkB-1f and alkB-1r (Kloos et al. 2006) in a
total volume of 30 ll containing: 1 ll DNA, 0Á5 lmol
l
À1 of each forward and reverse primer, 0Á2 mmol l
À1
dNTPs, 19 phire reaction buffer and 0Á6 ll of Phire Hot
Start II DNA Polymerase (Thermo Scientific). The PCR
conditions used were as follows: an initial denaturation at
98°C for 30 s, followed by 35 cycles of (98°C for 15 s,
55°C for 15 s, 72°C for 10 s) and a final extension at
72°C for 1 min. All amplicons were visualized on 1%
agarose gel.
Biosurfactant production and emulsification test
Two distinct methods were used for the screening of the
biosurfactant production by isolates: (i) the drop collapse
test and (ii) the oil spreading method. The drop collapse
test was performed by adding 7 ll of crude oil into each
well of a 96-well microtitre plate lid. The lid was equilibrated for 1 h at room temperature, and then 20 ll of
the culture was added to the surface of oil. After 1 min
of incubation, the shape of the drop on the surface of the
oil was observed. If the drop collapses, it indicates the
presence of surfactant (positive result), if it remains
beaded, it indicates the absence of surfactant (negative
response). Sodium dodecyl sulphate with the sterile distilled water was used as positive and negative control
respectively (Bodour and Miller-Maier 1998; Mahjoubi
et al. 2013). In the oil spreading method, 20 ml of distilled water was added to an empty Petri dish (90 mm),
and 10 ll of crude oil was added to the surface of water
to form a thin layer of hydrocarbons on the surface. The
following step was to apply 10 ll of bacterial culture on
the oil surface. The diameter of clear zone around the
bacterial suspension was measured as the activity of surfactants (Cappello et al. 2016). The emulsification index
(E24) of different bacterial culture was determined by
adding 2 ml of hexadecane to the same amount of bacterial culture. The mixture was vigorously mixed (vortex)
for 2 min. The mixture could stand for 24 h prior to
measurement. The emulsification activity was calculated
as a percentage of the height of the emulsified layer
divided by the total height of the liquid column (Hassanshahian et al. 2012).
Results
Toxicity assay
The bioluminescence inhibition assay (Microtox) based
on the Gram-negative bacterium V. fisheri was used to
establish the toxicity level of the polluted marine sediment. The data revealed that all samples inhibited the
bioluminescence of V. fisheri more than 20% and their
Journal of Applied Microbiology 126, 780--795 © 2018 The Society for Applied Microbiology
784
The use of oil-degrading bacteria as an alternative for environmental remediation (bioremediation)
N. Djahnit et al.