for biotechnological applications (Das and Chandran
2011). Interestingly, one isolate was reported for the first
time from oil-contaminated areas; this strain is related to
X. gelatinilytica. This Bacteroidetes was first time isolated
from seawater collected from India (Vaidya et al. 2015)
but has never been isolated from the Mediterranean Sea
nor hydrocarbon-contaminated environments. This strain
is closely related to the genus Bizionia that was found coexisting with Alcanivorax and Marinobacter isolated from
contaminated sediment samples (Chen et al. 2017).
Hydrocarbon-degrading bacteria were isolated from a
contaminated site in Tunisia and the dominant bacterial
group was also Proteobacteria but belonging to different
genera such as Acinetobacter and Stenotrophomons
(Mahjoubi et al. 2013). Our results were similar to those
obtained by Tapilatu et al. (2010) who isolated hydrocarbon-degrading bacteria from marine sediment from the
French coast (North western Mediterranean), and found
that the majority of isolates belonged to Alcanivorax. In
the same study, alkane-degrading bacteria belonging to
the genera Pseudomonas and Marinobacter were also isolated. Catania et al. (2015) studied the diversity of culturable hydrocarbon-degrading bacteria in Priolo bay
(Ionian Sea, Central Mediterranean) and found that they
were affiliated mainly to genera of obligate hydrocarbonoclastic bacteria (Alcanivorax) and/or to generalist hydrocarbon degraders (Thalassospira, Oleibacter, Vibrio,
Marinobacter, Rhodococcus, Pseudomonas) and that most
sediment and water samples were dominated by bacteria
related to Alcanivorax and Marinobacter genera. Our
results of microbial isolation and identification corroborated literature data about the presence of aerobic bacteria in petroleum-contaminated seawater and sediment
samples support the hypothesis that these bacteria might
play a role in oil biodegradation processes in situ.
Biosurfactants are a heterogeneous group of surfaceactive compounds produced by a wide variety of microorganisms. Surfactants enhance solubilization and removal
of contaminants (Batista et al. 2006). The action mechanism of biosurfactants lies in their accumulation at the
interface of immiscible compound, reducing the surface
tension and thereby increasing their surface area; this leads
to a higher bioavailability that eases the degradation of pollutants. Our results showed that there is a positive correlation between biosurfactant production and emulsification
activity. Isolates affiliated to Alcanivorax, Marinobacter and
Erythrobacter were the most efficient; at the same time they
represented the highest rates of crude oil degradation
(more than 50% of crude oil degraded). A relationship
between the levels of biosurfactant production and crude
oil biodegradation was observed (Hassanshahian et al.
2012). Strains producing high levels of biosurfactant can
better degrade crude oil, indicating that the production of
extracellular biosurfactants may be one of the underlying
mechanisms implemented by the isolates for crude oil
metabolization (Banat et al. 2010). But this suggestion was
not applicable for all studied strains. Indeed, Gordonia was
able to degrade 40% of crude oil after 1 week of incubation
but was not efficient in biosurfactant production. A different mechanism of direct contact to the n-alkanes by cell
adherence has been described for the Corynebacterium/
Mycobacterium/Nocardia complex to which Gordonia
belongs (Lo Piccolo et al. 2011).
In this investigation, we also studied the capacity of
selected strains to grow in the presence of various aliphatic and aromatic hydrocarbons as the sole carbon and
energy source. It was noted that most of strains tested
were able to metabolize aliphatic hydrocarbons, while the
aromatic fraction was metabolized by only five strains.
Aromatic hydrocarbons are more resistant against
biodegradation than aliphatic compounds; they often
cause serious problems during bioremediation (Rajaei
et al. 2013). Some strains were able to grow on both fractions.
It is uncommon to find micro-organisms having the
capacity to degrade both aliphatic and aromatic compounds. The degradation of the two types of hydrocarbons requires different metabolic pathways (M’rassi et al.
2015). Monooxygenase genes have been used as functional biomarkers for the characterization of aerobic
degrading bacterial populations in environmental samples
and in bioremediation experiments (Van Beilen et al.
2003). The specific primers of alkB, tol, xylA genes and
catabolic genes involved in hydrocarbon degradation were
used to detect the catabolic pathways of bacterial isolates.
The PCR amplification trials revealed the presence of
alkB gene in several bacterial strains. The xylA gene
involved in the aromatic hydrocarbon degradation pathway (BTEX degradation) was found in only one bacterial
isolate (PSF-43Sw). Strain PSF-43Sw related to M.
nitratireducens possesses both aliphatic and aromatic catabolic pathways, which confirms the ability of this strain
to metabolize both alkanes and aromatic hydrocarbons as
revealed also by the hydrocarbon utilization profile analysis. Some isolates growing in the presence of crude oil
gave no amplification product implying the existence of
other biodegradation pathways or highly divergent gene
sequences that suggest they probably harbour other
hydrocarbon-degrading genes (as AlmA, P450. . .)
(M’rassi et al. 2015).
The results obtained in this work demonstrate the
diversity of hydrocarbon-degrading bacteria from marinecontaminated area in Algeria, and their variable biodegradation abilities.
Knowledge of biodegradation potential and studies of
the biodegrading microbial communities associated with
Journal of Applied Microbiology 126, 780--795 © 2018 The Society for Applied Microbiology
792
The use of oil-degrading bacteria as an alternative for environmental remediation (bioremediation)
N. Djahnit et al.
2011). Interestingly, one isolate was reported for the first
time from oil-contaminated areas; this strain is related to
X. gelatinilytica. This Bacteroidetes was first time isolated
from seawater collected from India (Vaidya et al. 2015)
but has never been isolated from the Mediterranean Sea
nor hydrocarbon-contaminated environments. This strain
is closely related to the genus Bizionia that was found coexisting with Alcanivorax and Marinobacter isolated from
contaminated sediment samples (Chen et al. 2017).
Hydrocarbon-degrading bacteria were isolated from a
contaminated site in Tunisia and the dominant bacterial
group was also Proteobacteria but belonging to different
genera such as Acinetobacter and Stenotrophomons
(Mahjoubi et al. 2013). Our results were similar to those
obtained by Tapilatu et al. (2010) who isolated hydrocarbon-degrading bacteria from marine sediment from the
French coast (North western Mediterranean), and found
that the majority of isolates belonged to Alcanivorax. In
the same study, alkane-degrading bacteria belonging to
the genera Pseudomonas and Marinobacter were also isolated. Catania et al. (2015) studied the diversity of culturable hydrocarbon-degrading bacteria in Priolo bay
(Ionian Sea, Central Mediterranean) and found that they
were affiliated mainly to genera of obligate hydrocarbonoclastic bacteria (Alcanivorax) and/or to generalist hydrocarbon degraders (Thalassospira, Oleibacter, Vibrio,
Marinobacter, Rhodococcus, Pseudomonas) and that most
sediment and water samples were dominated by bacteria
related to Alcanivorax and Marinobacter genera. Our
results of microbial isolation and identification corroborated literature data about the presence of aerobic bacteria in petroleum-contaminated seawater and sediment
samples support the hypothesis that these bacteria might
play a role in oil biodegradation processes in situ.
Biosurfactants are a heterogeneous group of surfaceactive compounds produced by a wide variety of microorganisms. Surfactants enhance solubilization and removal
of contaminants (Batista et al. 2006). The action mechanism of biosurfactants lies in their accumulation at the
interface of immiscible compound, reducing the surface
tension and thereby increasing their surface area; this leads
to a higher bioavailability that eases the degradation of pollutants. Our results showed that there is a positive correlation between biosurfactant production and emulsification
activity. Isolates affiliated to Alcanivorax, Marinobacter and
Erythrobacter were the most efficient; at the same time they
represented the highest rates of crude oil degradation
(more than 50% of crude oil degraded). A relationship
between the levels of biosurfactant production and crude
oil biodegradation was observed (Hassanshahian et al.
2012). Strains producing high levels of biosurfactant can
better degrade crude oil, indicating that the production of
extracellular biosurfactants may be one of the underlying
mechanisms implemented by the isolates for crude oil
metabolization (Banat et al. 2010). But this suggestion was
not applicable for all studied strains. Indeed, Gordonia was
able to degrade 40% of crude oil after 1 week of incubation
but was not efficient in biosurfactant production. A different mechanism of direct contact to the n-alkanes by cell
adherence has been described for the Corynebacterium/
Mycobacterium/Nocardia complex to which Gordonia
belongs (Lo Piccolo et al. 2011).
In this investigation, we also studied the capacity of
selected strains to grow in the presence of various aliphatic and aromatic hydrocarbons as the sole carbon and
energy source. It was noted that most of strains tested
were able to metabolize aliphatic hydrocarbons, while the
aromatic fraction was metabolized by only five strains.
Aromatic hydrocarbons are more resistant against
biodegradation than aliphatic compounds; they often
cause serious problems during bioremediation (Rajaei
et al. 2013). Some strains were able to grow on both fractions.
It is uncommon to find micro-organisms having the
capacity to degrade both aliphatic and aromatic compounds. The degradation of the two types of hydrocarbons requires different metabolic pathways (M’rassi et al.
2015). Monooxygenase genes have been used as functional biomarkers for the characterization of aerobic
degrading bacterial populations in environmental samples
and in bioremediation experiments (Van Beilen et al.
2003). The specific primers of alkB, tol, xylA genes and
catabolic genes involved in hydrocarbon degradation were
used to detect the catabolic pathways of bacterial isolates.
The PCR amplification trials revealed the presence of
alkB gene in several bacterial strains. The xylA gene
involved in the aromatic hydrocarbon degradation pathway (BTEX degradation) was found in only one bacterial
isolate (PSF-43Sw). Strain PSF-43Sw related to M.
nitratireducens possesses both aliphatic and aromatic catabolic pathways, which confirms the ability of this strain
to metabolize both alkanes and aromatic hydrocarbons as
revealed also by the hydrocarbon utilization profile analysis. Some isolates growing in the presence of crude oil
gave no amplification product implying the existence of
other biodegradation pathways or highly divergent gene
sequences that suggest they probably harbour other
hydrocarbon-degrading genes (as AlmA, P450. . .)
(M’rassi et al. 2015).
The results obtained in this work demonstrate the
diversity of hydrocarbon-degrading bacteria from marinecontaminated area in Algeria, and their variable biodegradation abilities.
Knowledge of biodegradation potential and studies of
the biodegrading microbial communities associated with
Journal of Applied Microbiology 126, 780--795 © 2018 The Society for Applied Microbiology
792
The use of oil-degrading bacteria as an alternative for environmental remediation (bioremediation)
N. Djahnit et al.