Bacterial abundance
The bacterial abundance was measured only for seawater
samples by direct counting under epifluorescence as previously reported (Porter and Feig 1980; Catania et al.
2015). Volumes of seawater samples were fixed with
formaldehyde (2% v/v) then filtered on polycarbonate
black filters (0Á2 lm). The filters were stained with 4,6diamidino-2-phenylindole (DAPI; Sigma Aldrich S.R.L.,
Milan, Italy). Slides were examined under a Zeiss Axioplan 2 Imaging epifluorescence microscope (Zeiss; Carl
Zeiss Inc., Thornwood, NY). Results were expressed as
number of cells per millilitre of seawater (Cappello et al.
2007).
DNA extraction
A volume of 100 ml of collected seawater from different
sampling stations was filtered on 0Á2 lm filter (Millipore,
Milan, Italy). Then filters were used for extraction of total
DNA using the MasterPure
TM complete DNA and RNA
purification kit (Epicentre, Madison, WI). Total DNA
was extracted from marine sediment samples using the
FastDNA
TM SPIN Kit for Soil (MP Biomedicals, Santa
Ana, CA), according to the supplier’s specifications as
described previously (Catania et al. 2015). The concentration and purity of total DNA extracted from samples
were determined using NanoDROP ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA). DNAs
were stored at À20°C until further analysis.
Denaturing gradient gel electrophoresis analysis
The variable region V3 corresponding to positions 341–
534 in the Escherichia coli 16S rRNA sequence was amplified by PCR from total genomic DNA using primers
341f-GC and 534r (Muyzer et al. 1993) as described in
Catania et al. (2016). DGGE profiles were visually analysed and the relative position of each band was recorded
in a resulting 0–1 matrix that was put into the PAST software package and analysed using multivariate cluster
analysis based on Jaccard dissimilarity. Some dominant
DGGE bands were excised with a sterile pipette tip and
resuspended overnight in 20 ll DNA/RNA free water
(GIBCO, Milano, Italy) at 4°C and then frozen at À20°C.
Sediment and seawater samples from station 5 are missed
due to technical problems.
Enrichment and isolation of aerobic hydrocarbondegrading bacteria
To isolate autochthonous hydrocarbon-degrading bacteria
and to investigate their degradation potential, enrichment
cultures were set up. Aliquots of sediment and seawater
were transferred to sterile flasks containing 100 ml of
ONR7a supplemented respectively with 0Á1% (v/v) crude
oil, n-alkane mixture (C 14 +C 16 +C 18 , 1 : 1 : 1) or a mixture of benzene/toluene/xylene (1 : 1 : 1). Cultures were
incubated at 25°C with shaking for 10 days. Then aliquots were removed and placed in fresh medium ONR7a
containing the same concentration of hydrocarbons and
incubated under the same conditions. Subcultures were
spread onto plates containing solid ONR7a medium with
same hydrocarbons supplied on sterile filter paper on the
lid of the Petri dish with the same concentrations as
mentioned previously. Plates were incubated at 25°C.
Phenotypically different colonies obtained from plates
were purified and transferred to fresh liquid medium.
Only isolates exhibiting pronounced growth on crude oil
and/or other hydrocarbons were stored in 20% glycerol
solution at À20°C for further characterization (Hassanshahian et al. 2012; Catania et al. 2016).
Taxonomical analysis of 16S rRNA genes and
sequencing
Molecular characterization of isolates was based on 16S ribosomal RNA (rRNA) gene sequence analysis. The bacterial
DNA was extracted using colony PCR method (China et al.
1996). PCR amplification of the 16S rRNA gene was
performed using the domain-specific forward primer
Bac27_F (AGAGTTTGATCCTGGCTAG) and the universal
reverse primer Uni_1492R (TACGYTACCTTGTTAC
GACTT) (Lane 1991). The amplification reaction was carried
out in a total volume of 30 ll consisting of 1 ll DNA (between 10 and 100 ng), 0Á2 lmol l
À1 of each forward and
reverse primer, 0Á2 mmol l
À1 dNTPs, 19 reaction buffer
and 0Á5 U of Taq DNA polymerase (Qiagen, Milano, Italy).
PCR was carried out in a Mastercycler Gradient (Eppendorf,
Hamburg, Germany) using the following cycles programme:
one cycle at 94°C for 30 s; 30 cycles of (94°C for 30 s, 50°C
for 60 s and 68°C for 30 s) and a final extension cycle at
68°C for 5 min. PCR amplicons were analysed in 1% agarose
gel electrophoresis. The expected amplicon size was around
1450 bp. The amplified 16S rDNA fragments were sequenced
using Macrogen Europe Service (Amsterdam, The Netherlands). The sequences were analysed using the BLAST tool of
the National Centre for Biotechnology Information website
(www.ncbi.nlm.nih.gov/BLAST/) (Altschul et al. 1990). 16S
rRNA sequences were deposited in Genbank under accession
number from MH425630 to MH425652.
Hydrocarbon utilization profile and degradation abilities
To determine the use of different hydrocarbons as carbon
source by the bacterial strains, each strain was inoculated
Journal of Applied Microbiology 126, 780--795 © 2018 The Society for Applied Microbiology
783
N. Djahnit et al.
The use of oil-degrading bacteria as an alternative for environmental remediation (bioremediation)
The bacterial abundance was measured only for seawater
samples by direct counting under epifluorescence as previously reported (Porter and Feig 1980; Catania et al.
2015). Volumes of seawater samples were fixed with
formaldehyde (2% v/v) then filtered on polycarbonate
black filters (0Á2 lm). The filters were stained with 4,6diamidino-2-phenylindole (DAPI; Sigma Aldrich S.R.L.,
Milan, Italy). Slides were examined under a Zeiss Axioplan 2 Imaging epifluorescence microscope (Zeiss; Carl
Zeiss Inc., Thornwood, NY). Results were expressed as
number of cells per millilitre of seawater (Cappello et al.
2007).
DNA extraction
A volume of 100 ml of collected seawater from different
sampling stations was filtered on 0Á2 lm filter (Millipore,
Milan, Italy). Then filters were used for extraction of total
DNA using the MasterPure
TM complete DNA and RNA
purification kit (Epicentre, Madison, WI). Total DNA
was extracted from marine sediment samples using the
FastDNA
TM SPIN Kit for Soil (MP Biomedicals, Santa
Ana, CA), according to the supplier’s specifications as
described previously (Catania et al. 2015). The concentration and purity of total DNA extracted from samples
were determined using NanoDROP ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA). DNAs
were stored at À20°C until further analysis.
Denaturing gradient gel electrophoresis analysis
The variable region V3 corresponding to positions 341–
534 in the Escherichia coli 16S rRNA sequence was amplified by PCR from total genomic DNA using primers
341f-GC and 534r (Muyzer et al. 1993) as described in
Catania et al. (2016). DGGE profiles were visually analysed and the relative position of each band was recorded
in a resulting 0–1 matrix that was put into the PAST software package and analysed using multivariate cluster
analysis based on Jaccard dissimilarity. Some dominant
DGGE bands were excised with a sterile pipette tip and
resuspended overnight in 20 ll DNA/RNA free water
(GIBCO, Milano, Italy) at 4°C and then frozen at À20°C.
Sediment and seawater samples from station 5 are missed
due to technical problems.
Enrichment and isolation of aerobic hydrocarbondegrading bacteria
To isolate autochthonous hydrocarbon-degrading bacteria
and to investigate their degradation potential, enrichment
cultures were set up. Aliquots of sediment and seawater
were transferred to sterile flasks containing 100 ml of
ONR7a supplemented respectively with 0Á1% (v/v) crude
oil, n-alkane mixture (C 14 +C 16 +C 18 , 1 : 1 : 1) or a mixture of benzene/toluene/xylene (1 : 1 : 1). Cultures were
incubated at 25°C with shaking for 10 days. Then aliquots were removed and placed in fresh medium ONR7a
containing the same concentration of hydrocarbons and
incubated under the same conditions. Subcultures were
spread onto plates containing solid ONR7a medium with
same hydrocarbons supplied on sterile filter paper on the
lid of the Petri dish with the same concentrations as
mentioned previously. Plates were incubated at 25°C.
Phenotypically different colonies obtained from plates
were purified and transferred to fresh liquid medium.
Only isolates exhibiting pronounced growth on crude oil
and/or other hydrocarbons were stored in 20% glycerol
solution at À20°C for further characterization (Hassanshahian et al. 2012; Catania et al. 2016).
Taxonomical analysis of 16S rRNA genes and
sequencing
Molecular characterization of isolates was based on 16S ribosomal RNA (rRNA) gene sequence analysis. The bacterial
DNA was extracted using colony PCR method (China et al.
1996). PCR amplification of the 16S rRNA gene was
performed using the domain-specific forward primer
Bac27_F (AGAGTTTGATCCTGGCTAG) and the universal
reverse primer Uni_1492R (TACGYTACCTTGTTAC
GACTT) (Lane 1991). The amplification reaction was carried
out in a total volume of 30 ll consisting of 1 ll DNA (between 10 and 100 ng), 0Á2 lmol l
À1 of each forward and
reverse primer, 0Á2 mmol l
À1 dNTPs, 19 reaction buffer
and 0Á5 U of Taq DNA polymerase (Qiagen, Milano, Italy).
PCR was carried out in a Mastercycler Gradient (Eppendorf,
Hamburg, Germany) using the following cycles programme:
one cycle at 94°C for 30 s; 30 cycles of (94°C for 30 s, 50°C
for 60 s and 68°C for 30 s) and a final extension cycle at
68°C for 5 min. PCR amplicons were analysed in 1% agarose
gel electrophoresis. The expected amplicon size was around
1450 bp. The amplified 16S rDNA fragments were sequenced
using Macrogen Europe Service (Amsterdam, The Netherlands). The sequences were analysed using the BLAST tool of
the National Centre for Biotechnology Information website
(www.ncbi.nlm.nih.gov/BLAST/) (Altschul et al. 1990). 16S
rRNA sequences were deposited in Genbank under accession
number from MH425630 to MH425652.
Hydrocarbon utilization profile and degradation abilities
To determine the use of different hydrocarbons as carbon
source by the bacterial strains, each strain was inoculated
Journal of Applied Microbiology 126, 780--795 © 2018 The Society for Applied Microbiology
783
N. Djahnit et al.
The use of oil-degrading bacteria as an alternative for environmental remediation (bioremediation)