containing alkB included those affiliated to Alcanivorax,
Marinobacter, Gordonia, Pseudomonas and Brevibacterium.
At least one isolate from each genus was selected for
sequencing (Table 5). Nucleotide sequence of alkB
revealed that identity of the amplified fragments was
coherent with the 16S-based identification for Alcanivorax, Marinobacter and Gordonia at genus level, while the
alkB gene belonging to Brevibacterium showed 99%
nucleotide sequence identity with Dietzia sp. alkB gene.
In contrast, alkB could not be detected in isolates related
to Halomonas, Xanthomarina, Erythrobacter and Micrococcus although they were able to grow on n-alkanes. Strains
that were able to grow on aromatic hydrocarbons (PSF14Sw, PSF-16Sw, PSF-31S, PSF-35Sw and PSF-43Sw)
were examined for the presence of xylene monooxygenase
gene by PCR. A PCR product was obtained only for
strain PSF-43Sw with primers XYLA-F/XYLA-R. The
PSF-43Sw xylA nucleotide sequence is identical to that of
Marinobacter sp. which confirms the results obtained by
16S and alkB sequencing for this isolate.
Biosurfactant production and emulsification activity
In this study, we tested the production of biosurfactant
by different methods: the drop collapse test, the oil
spreading test and the emulsification index E24 were
performed for each strain. The results are shown in
Table 4. Among the 23 strains screened, 16 (69Á6%)
strains were positive for drop collapse activity. All these
strains are related to genus Alcanivorax and Marinobacter; one strain is related to Erythrobacter and one strain
to Brevibacterium. Diameter of clearing on the oil surface varied between 2 and 6 mm. The highly producer
species that showed 6 mm in the oil spreading test are
PSF-31S and PSF-12Sw related to M. hydrocarbonoclasticus and A. borkumensis respectively. Oil spreading assay
results were in corroboration with those obtained by
drop collapse assay. Strains found with positive drop
collapse results represented the highest diameter of
clearing zone. Except for strain PSF-23S related to
Micrococcus yunnanensis that was positive for drop collapse activity but showed the lowest size of clearing
zone (2 mm). Emulsification index (E24) determines the
productivity of bioemulsifier and is given as a percentage of the height of the emulsified layer divided by the
total height of the liquid column. The major parts of
organisms screened were found to produce biosurfactant
by their ability to emulsify hexadecane. In the subclass
of Alphaproteobacteria, E. citreus (strain SF-14Sw)
exhibited the highest emulsification index with 49% of
activity, while A. borkumensis (Strain PSF-12Sw) proved
to be the most active strain among the fraction
Gammaproteobacteria with 52% of the emulsification.
No emulsification activity was observed for X. gelatinilytica (PSF-8Sw). Emulsification activity was also shown
to be important (40%) for the Gram-positive bacteria
Brevibacterium celere (PSF-5Sw), which demonstrated
also a positive result for the drop collapse test. The
0
20
40
60
80
100
120
A C
P S F - 5 s w
P S F - 6 s
P S F - 2 3 s
P S F - 8 s w
P S F - 1 4 s w
P S F - 1 6 s w
P S F - 1 2 s w
P S F - 3 0 s w
P S F - 1 3 s w
P S F - 1 5 s w P S F - 3 1 s P S F - 4 3 s w
P S F - 3 5 s w
P S F - 2 1 s w
% of remaining crude oil
Figure 4 Degradation capability of the bacteria expressed as percentage of oil remaining after cultivation in ONR7a with 0Á1% of crude oil
(30 Æ 1°C for 1 week on rotator shaker, 180 g). AC: abiotic control. The names of the isolates are reported in Table 4.
Journal of Applied Microbiology 126, 780--795 © 2018 The Society for Applied Microbiology
789
N. Djahnit et al.
The use of oil-degrading bacteria as an alternative for environmental remediation (bioremediation)
Marinobacter, Gordonia, Pseudomonas and Brevibacterium.
At least one isolate from each genus was selected for
sequencing (Table 5). Nucleotide sequence of alkB
revealed that identity of the amplified fragments was
coherent with the 16S-based identification for Alcanivorax, Marinobacter and Gordonia at genus level, while the
alkB gene belonging to Brevibacterium showed 99%
nucleotide sequence identity with Dietzia sp. alkB gene.
In contrast, alkB could not be detected in isolates related
to Halomonas, Xanthomarina, Erythrobacter and Micrococcus although they were able to grow on n-alkanes. Strains
that were able to grow on aromatic hydrocarbons (PSF14Sw, PSF-16Sw, PSF-31S, PSF-35Sw and PSF-43Sw)
were examined for the presence of xylene monooxygenase
gene by PCR. A PCR product was obtained only for
strain PSF-43Sw with primers XYLA-F/XYLA-R. The
PSF-43Sw xylA nucleotide sequence is identical to that of
Marinobacter sp. which confirms the results obtained by
16S and alkB sequencing for this isolate.
Biosurfactant production and emulsification activity
In this study, we tested the production of biosurfactant
by different methods: the drop collapse test, the oil
spreading test and the emulsification index E24 were
performed for each strain. The results are shown in
Table 4. Among the 23 strains screened, 16 (69Á6%)
strains were positive for drop collapse activity. All these
strains are related to genus Alcanivorax and Marinobacter; one strain is related to Erythrobacter and one strain
to Brevibacterium. Diameter of clearing on the oil surface varied between 2 and 6 mm. The highly producer
species that showed 6 mm in the oil spreading test are
PSF-31S and PSF-12Sw related to M. hydrocarbonoclasticus and A. borkumensis respectively. Oil spreading assay
results were in corroboration with those obtained by
drop collapse assay. Strains found with positive drop
collapse results represented the highest diameter of
clearing zone. Except for strain PSF-23S related to
Micrococcus yunnanensis that was positive for drop collapse activity but showed the lowest size of clearing
zone (2 mm). Emulsification index (E24) determines the
productivity of bioemulsifier and is given as a percentage of the height of the emulsified layer divided by the
total height of the liquid column. The major parts of
organisms screened were found to produce biosurfactant
by their ability to emulsify hexadecane. In the subclass
of Alphaproteobacteria, E. citreus (strain SF-14Sw)
exhibited the highest emulsification index with 49% of
activity, while A. borkumensis (Strain PSF-12Sw) proved
to be the most active strain among the fraction
Gammaproteobacteria with 52% of the emulsification.
No emulsification activity was observed for X. gelatinilytica (PSF-8Sw). Emulsification activity was also shown
to be important (40%) for the Gram-positive bacteria
Brevibacterium celere (PSF-5Sw), which demonstrated
also a positive result for the drop collapse test. The
0
20
40
60
80
100
120
A C
P S F - 5 s w
P S F - 6 s
P S F - 2 3 s
P S F - 8 s w
P S F - 1 4 s w
P S F - 1 6 s w
P S F - 1 2 s w
P S F - 3 0 s w
P S F - 1 3 s w
P S F - 1 5 s w P S F - 3 1 s P S F - 4 3 s w
P S F - 3 5 s w
P S F - 2 1 s w
% of remaining crude oil
Figure 4 Degradation capability of the bacteria expressed as percentage of oil remaining after cultivation in ONR7a with 0Á1% of crude oil
(30 Æ 1°C for 1 week on rotator shaker, 180 g). AC: abiotic control. The names of the isolates are reported in Table 4.
Journal of Applied Microbiology 126, 780--795 © 2018 The Society for Applied Microbiology
789
N. Djahnit et al.
The use of oil-degrading bacteria as an alternative for environmental remediation (bioremediation)