MARINE AND FISHERY SCIENCES 34 (1): 21-36 (2021)
https://doi.org/10.47193/mafis.3412021010304
ABSTRACT. Hydrocarbon degrading bacteria (HDB) were monitored since 2006 to 2018 at the
‘Estación Permanente de Estudios Ambientales’ (EPEA), in order to analyze its abundance and the
potentiality to metabolize these pollutants. The presence of HDB was detected with counts values
ranging between 103and 105UFC ml-1. A slight increase was observed over time, which could be
linked to changes in marine temperature reported within the last years. Thirty-six HDB were tested
for growth on various hydrocarbons and some of them showed a broad biodegradation profile.
Moreover, from phenanthrene (Phe) enrichment cultures, five strains were phylogenetically identi-
fied as Halomonas sp. E1, E2 and E3; Rhodococcus sp. E4 and Pseudomonas sp. E5. Complete Phe
degradation was demonstrated for E4 and E5 strains, while E1, E2, E3 and E4 strains displayed sur-
factant production. This study contributed with the first knowledge about the intrinsic hydrocarbon
biodegradation potential by bacterial communities at EPEA. Some of the strains exhibited physio-
logical properties that might have ecological significance on environmental alterations as the pres-
ence of pollutants. Particularly, Rhodococcus sp. E4 could be an alternative for microbial selection
in the degradation of polycyclic aromatic hydrocarbons. Further studies are needed to evaluate the
impact of the climate change on microbial-mediated detoxification processes.
Key words: PAH, bioremediation, biosurfactant.
Caracterización de bacterias degradadoras de hidrocarburos en la estación EPEA, costa del
Atlántico Sur
RESUMEN. Las bacterias degradadoras de hidrocarburos (BDH) fueron monitoreadas desde 2006
a 2018 en la Estación Permanente de Estudios Ambientales (EPEA), con el fin de analizar su abun-
dancia y la potencialidad de metabolizar estos contaminantes. La presencia de BDH se detectó con
valores de recuento que oscilaron entre 103y 105UFC ml-1. Se observó un ligero aumento a lo largo
del tiempo, que podría estar relacionado con cambios en la temperatura marina reportados en los últi-
mos años. Se analizaron 36 BDH para determinar su crecimiento en varios hidrocarburos y algunas de
ellas mostraron un perfil de biodegradación amplio. Además, a partir de cultivos de enriquecimiento
con fenantreno (Phe), se identificaron filogenéticamente cinco cepas como Halomonas sp. E1, E2 y
E3; Rhodococcus sp. E4 y Pseudomonas sp. E5. Se demostró una degradación completa de Phe para
las cepas E4 y E5, mientras que las cepas E1, E2, E3 y E4 mostraron producción de surfactante. Este
estudio contribuyó con el primer conocimiento sobre el potencial intrínseco de biodegradación de los
hidrocarburos por las comunidades bacterianas en EPEA. Algunas de las cepas exhibieron propieda-
des fisiológicas que pueden tener importancia ecológica sobre alteraciones ambientales como la pre-
sencia de contaminantes. En particular, Rhodococcus sp. E4 podría ser una alternativa para la selec-
ción microbiana en la degradación de hidrocarburos poliaromáticos. Se necesitan más estudios para
evaluar el impacto del cambio climático en los procesos de desintoxicación mediados por microbios.
Palabras clave: PAH, biorremediación, biosurfactante.
21
*Correspondence:
silviap_ar@inidep.edu.ar
Received: 29 October 2020
Accepted: 15 December 2020
ISSN 2683-7595 (print)
ISSN 2683-7951 (online)
https://ojs.inidep.edu.ar
Journal of the Instituto Nacional de
Investigación y Desarrollo Pesquero
(INIDEP)
This work is licensed under a Creative
Commons Attribution-
NonCommercial-ShareAlike 4.0
International License
Marine and
Fishery Sciences
MAFIS
ORIGINAL RESEARCH
Characterization of hydrocarbon degrading bacteria at EPEA station,
South Atlantic coast
SILVIA R. PERESSUTTI*
Instituto Nacional de Investigación y Desarrollo Pesquero (INIDEP), Paseo Victoria Ocampo Nº 1, Escollera Norte,
B7602HSA - Mar del Plata, Argentina
INTRODUCTION
Despite the flow of water masses and the dilu-
tion power of marine waters, oil pollution caused
by industrial and vessel activities has a significant
impact on these systems. It is broadly recognized
that hydrocarbons contamination has damaged
oceans, seas and coastal zones and represents a
continuous threat to the marine environment sus-
tainability (McGenity et al. 2012). Main sources of
oil pollution in open oceans and coastal waters
occur by accidental spills and deliberate discharge
of ballast, wash waters from oil tankers, and bilge
waste discharges, producing contamination and
severe adverse effects on the ecosystem (Etkin
2010). Normal shipping operations account for
over 70% of the hydrocarbons entering the sea
from marine transportation (Nievas et al. 2006).
According to the International Maritime Organiza-
tion (IMO), the shipping industry that fulfils more
than 90% of trade across the world with the help
of around 90,000 marine vessels contributes heav-
ily to global pollution and climate change (Jäger-
brand et al. 2019). Likewise, marine environments
are especially vulnerable to oil spills because they
are poorly contained and difficult to mitigate.
As a result of oil contamination in marine
ecosystems, adverse effects have been observed on
aquatic organisms at sub-lethal concentrations
(McGentity et al. 2012). Hydrocarbons can
become dangerous fundamentally in the event that
they enter the food chain, since several compounds
as polycyclic aromatic hydrocarbons (PAHs) are
toxic, mutagenic and carcinogenic (Perelo 2010).
Hydrocarbons have also a natural potential for
bioaccumulation in marine organisms with possi-
ble transfer to humans via seafood and are there-
fore considered as substances of potential human
health hazards (Mrozik et al. 2003).
Biodegradation by natural microbial popula-
tions is the most basic and reliable mechanism by
which thousands of xenobiotic pollutants, e.g.,
hydrocarbons, are removed from the environment
(Cappello et al. 2007). Microbial communities not
only play a central role in the main biogeochemical
cycles but also in the global recycling of pollutants
(Falcón et al. 2008). Autochthonous hydrocarbon-
degrading microorganisms living in marine sys-
tems would be better adapted to restore the hydro-
carbon contamination in seawater. In general, bac-
teria have great adaptability to diverse environ-
mental conditions, fast population growth and
metabolic versatility (Deng et al. 2014). Therefore,
the understanding of the microbial community and
its catabolic activity are essential for the assess-
ment of its biodegradation potential and for the
effective remediation of contaminated areas
(Muangchinda et al. 2015; Shi et al. 2019). These
studies are also important in the context of the cli-
mate change. Despite the importance of microbes
in the process of global recycling of anthropogenic
pollutants, the potential interactions of ocean acid-
ification, UVR, temperature, anthropogenic pollu-
tants, and marine microbial communities have
been largely ignored. It has recently been demon-
strated that such interactions could alter microbial-
mediated detoxification processes (Coelho et al.
2013; Louvado et al. 2018; Cabral et al. 2019).
The permanent coastal station EPEA (Estación
Permanente de Estudios Ambientales) is one of
the foundation stations of ANTARES, a network
of time series stations along South America
(www.antares.ws) located in the coastal waters of
Argentina, 27 nautical miles south from Mar del
Plata harbor. The main objective of EPEA time-
series is to understand the annual and inter-annual
dynamics of environmental variables and all com-
ponents of plankton and follow possible long-term
changes. This station is affected by the increasing
vessel traffic causing oil pollution in the sea.
The aims of this research was to monitor the
hydrocarbon degrading bacteria (HDB), isolated
from EPEA station since 2006 to 2018, in order to
analyze its abundance over time and the poten-
tiality to metabolize these pollutants. Autochtho-
nous bacteria capable of degrading the polyaro-
22 MARINE AND FISHERY SCIENCES 34 (1): 21-36 (2021)
matic hydrocarbon phenanthrene (Phe) were also
selected and identified, characterizing their
biodegradation capacity and emulsifying activity.
Microorganisms of several genera like Rhodococ-
cus, Pseudomonas, Burkholderia, Sphingomonas,
Acinetobacter and Mycobacterium have been pre-
viously identified as PAH-degraders, and com-
plete PAH mineralization has been demonstrated
for both low- and high-molecular-weight PAHs
(Johnsen et al. 2005; Ghosal et al. 2016). In addi-
tion, some PAH-degrading bacteria display strate-
gies to improve hydrocarbon accessibility, such
as biosurfactant production (Pedetta et al. 2013).
MATERIALS AND METHODS
Study area and sampling
Sampling was performed during 46 research
cruises (Table 1) carried out by research vessels
(INIDEP) from 2006 to 2018 at the EPEA station,
located at 38° 28′ S and 57° 41′ W in the Atlantic
Ocean (Figure 1). Surface water samples were
collected with a bucket and transferred to sterile
plastic containers and stored at 4 °C. In order to
23
PERESSUTTI: HYDROCARBON DEGRADING BACTERIA AT EPEA STATION
Table 1. Salinity and temperature values during EPEA research cruises.
Research Salinity Temperature Month/ Research Salinity Temperature Month/
cruise (°C) year cruise (°C) year
CC0906 33.602 13.467 10/2006 EH0613 33.765 15.383 12/2013
CC1206 33.548 15.913 11/2006 OB0214 34.260 19.170 03/2014
OB0107 33.661 19.483 01/2007 AH0215 23.177 02/2015
CC0407 33.756 11.259 07/2007 AH0315 33.765 17.430 04/2015
CC0607 12.467 10/2007 AH0515 33.804 10.982 09/2015
OB0108 33.793 11.804 10/2008 AH0216 33.880 17.910 04/2016
OB0408 33.405 14.337 12/2008 AH0516 33.836 10.919 09/2016
OB0109 33.524 20.185 01/2009 AH0716 33.762 12.215 10/2016
CC0109 33.538 20.239 02/2009 EH0117 33.966 21.170 02/2017
OB0409 33.804 20.533 03/2009 AH0217 34.099 17.850 05/2017
OB0609 34.112 18.124 04/2009 AH0317 34.068 16.726 06/2017
CC0809 34.027 13.895 06/2009 AH0417 33.863 11.441 08/2017
CC0909 34.075 12.663 07/2009 AH0617 33.663 12.274 09/2017
CC1109 34.053 10.325 08/2009 AH0817 33.608 15.570 11/2017
CC0110 33.970 10.313 08/2010 EH0118 34.064 19.990 01/2018
CC0510 33.710 11.247 10/2010 VA0318 34.313 19.648 04/2018
CC1010 33.652 19.075 12/2010 AH0218 34.208 17.217 05/2018
CC0311 33.631 21.590 01/2011 AH0318 34.105 14.151 06/2018
OB0611 34.034 13.468 06/2011 AH0418 33.848 11.844 07/2018
OB0212 33.670 11.759 10/2012 VA1218 33.888 10.845 08/2018
OB0413 34.065 12.026 07/2013 AH0518 33.982 10.894 09/2018
CR0113 33.907 10.652 08/2013 VA1318 33.888 13.051 10/2018
OB0513 33.853 9.942 09/2013 AH0718 33.608 15.570 12/2018
describe the oceanography of the station, water
temperature (°C) and salinity were measured with
a CTD (Sea-Bird 19-01 CTD, SN 1268) by
BaRDO (Base Regional de Datos Oceanográfi-
cos) of the Instituto Nacional de Investigación y
Desarrollo Pesquero (INIDEP).
Bacterial abundance and isolation of hydro-
carbon degrading bacteria (HDB)
To determine bacterial counts and to isolate
pure bacterial strains, 3-fold serial dilutions were
performed in saline solution (NaCl 9%, p/v).
Duplicate aliquots from each dilution were
spread onto mineral salts medium (MSM) agar
plates (Schlegel et al. 1961), modified by the
addition of NaCl 3% (w/v) and 50 ml of diesel oil
as the sole carbon and energy source. MSM agar
plates with no carbon source were used as nega-
tive control. Plates were incubated aerobically at
25 °C for 7-14 days, and those yielding 30 to 300
colonies were afterwards directly counted and
expressed as CFU ml-1. Colonies with distinct
morphologies were picked and subsequently
purified by repetitive streaking onto diesel oil-
MSM agar. Pure cultures of final isolates selected
were preserved as 10% dimethyl sulfoxide
(DMSO) stocks at -80 °C.
Hydrocarbon utilization profile of the strains
Thirty-six purified strains selected from the
diesel oil-MSM agar plates were tested for growth
on various hydrocarbons used as sole carbon
source in order to investigate their degradation
potential as was reported previously (Peressutti et
al. 2003). Suspensions of the strains were inocu-
lated in MSM liquid medium supplemented with
technical hydrocarbon mixtures (diesel oil,
kerosene and mineral oil) and pure hydrocarbons
(n-pentane, n-hexane, n-octane, n-pentadecane, n-
hexadecane, dodecane, phenyldecane, cyclohexa-
ne, benzene, toluene, naphthalene, anthracene and
phenanthrene). Substrates were added at 0.5%
24 MARINE AND FISHERY SCIENCES 34 (1): 21-36 (2021)
Figure 1. Location of the ‘Estación Permanente de Estudios Ambientales’ (EPEA).
42°
41°
40°
39°
38°
37°
36°
35°
S
EPEA
W 61° 59° 57° 55° 53°
50 m
EG.: 38° 28′ S-57° 41′ W
54°56°58°60°62°
Buenos Aires
Argentina
Uruguay
(v/v), except for aromatic hydrocarbons which
were used at 0.1% (v/v or p/v) because higher
concentrations could be toxic for cellular growth.
Flasks were incubated at 25 °C for 14 days and
bacterial growth was evaluated by optical density
at 600 nm (OD600). Control cultures lacking a car-
bon source were performed for each isolate.
Degradation of PAH and biotic factors associ-
ated
Enrichment and isolation of PAH-degrading
strains
In order to study PAH-degrading bacteria,
enrichment cultures with Phe as the sole carbon
and energy source were set up according to Pedet-
ta et al. (2013). Water sub-samples (10 ml) were
inoculated into 500-ml flasks containing 100 ml
of MSM liquid medium supplemented with Phe
(160 mg l-1). Cultures were incubated aerobically
on an orbital shaker at 25 °C and 150 rpm for 14
days and bacterial growth was evaluated by opti-
cal density at 600 nm (OD600). Subsequently, 100
µl aliquots of cultures were spread onto MSM-
Phe agarose plates (Bogardt and Hemmingsen
1992), incubated at 25 °C for 7 days and colonies
of candidate Phe-degrading strain were picked up
and further purified by repetitive streaking on the
same fresh agar medium. Pure cultures of final
isolates selected were preserved as 10% DMSO
stocks at -80 °C.
Molecular identification of the bacterial isolates
Molecular identification was carried out by ana-
lyzing 16S rDNA gene sequencing. Genomic
DNA from each isolate was extracted according to
Wilson (2001), and its quality checked in a 0.8%
agarose gel electrophoresis after staining with
SyberSafe (Invitrogen, Argentina). PCR amplifi-
cations were performed by using the universal
primers F27 (5’-AGAGTTTGATCMTGGCT-
CAG-3’) and R1492 (5’-TACGGYTACCTTGT-
TACGACTT-3’) (Devereux and Willis 1995). The
reaction mixture contained: extracted DNA 1 µl,
GoTaq DNA polimerase (Promega) 5 UI/µl, buffer
5X, BSA 10 mg/ml, dNTPs 2.5 mM, each primer
0.5 μl and sterile double-distilled water was added
up to the end volume of 25 μl. The program used
for the amplification was: 5 min at 94 °C, 40
cycles of 30 s at 94 °C, 30 s at 58 °C, and 30 s at
72 °C; and a final elongation of 15 min at 72 °C
(Olivera et al. 2005), in a thermocycler (Life
Express, TC-96/T/H.a). PCR products were elec-
trophoresed on 1.5% (w/v) agarose gels containing
SyberSafe and visualized with UV in GelDoc EQ
(Bio-Rad, Hercules, CA, USA. DNA fragments of
expected size (1.3 kbp) were eluted, purified, and
sequenced commercially at INTA Castelar
(Argentina) by using the primers F63 (5’-
CAGGCCTAACACATGCAAGTC-3’) and F530
(5’-GTGCCAGCMGCCGCGG-3’). The resulting
sequences of the amplified fragments were com-
pared against sequences contained within Public
Database (NCBI/BLAST). Then, sequences were
analyzed phylogenetically with the MEGA 5.2
program (Tamura et al. 2011). Phylogenetic trees
were constructed through the neighbor-joining
(NJ) algorithm from a distance matrix calculated
following Tamura-Nei model plus discrete
Gamma distribution. Stability among the clades
was assessed with the 1,000-replication bootstrap
analysis. 16S rRNA sequences were deposited at
the GenBank database under accession numbers
MW160443 to MW160447.
Phenanthrene degradation
Biodegradation assays were conducted inocu-
lating an exponential phase culture of each bacte-
rial strain in 50 ml of MSM-Phe liquid medium,
and a non-inoculated flask was used as abiotic
control. Cultures were incubated aerobically on
an orbital shaker at 28 °C and 150 rpm for 12
days. To measure residual Phe concentrations, 3-
ml aliquots were withdrawn from the cultures
each 48 h and subsequently extracted with 6 ml of
acetonitrile. Tubes were incubated on an orbital
shaker for 1 h at 25 °C and 150 rpm. After that,
extracts of each culture were centrifuged (2500 g,
25
PERESSUTTI: HYDROCARBON DEGRADING BACTERIA AT EPEA STATION
10 min) and supernatants analyzed by reverse-
phase HPLC according to NIOSH (1998), at
CNEA (Buenos Aires). Chromatographic meas-
urements were carried out with an ACCELA 600
HPLC instrument (Thermo Scientific, USA),
consisting of a quaternary pump, an autosampler
and a photodiode-array detector. Column oven
temperature was set at 50 °C and quantification
wavelength was 254 nm. Separation was per-
formed using a 3 μm particle C-18 column of 250
×4.6 mm (Inertsil ODS-3; GL Science, Japan).
Isocratic elution with 80% acetonitrile/20% water
was performed at a flow rate of 0.9 ml min-1.
Biosurfactant production
Two distinct methods were used for the screen-
ing of the biosurfactant production by isolates: (i)
the drop collapse test and (ii) the emulsification
assay. The drop collapse test was performed
according to Jain et al. (1991) by adding 1 µl of
methylene blue [0.1% (w/v)] to 20 µl of cell-free
medium from saturated cultures grown in MSM-
Phe. The resulting mixture was spotted onto a
piece of Parafilm sheet (Pechiney Plastic Packag-
ing, USA), and after 5 min of incubation the
shape of the drop on the surface of the oil was
observed. If the drop collapsed, the presence of
surfactant (positive result) was indicated; if it
remained beaded, absence of surfactant (negative
response) was implied. Methylene blue was
added for visualization purposes without influ-
encing in droplet collapse activity. Fresh MSM-
Phe medium containing either no addition or 1%
sodium dodecyl sulphate was used as negative
and positive controls, respectively.
The emulsification index (E24) of the culture
supernatant was determined by adding 2 ml of
hexadecane to the same amount of aqueous super-
natant. The mixture was vigorously mixed (vor-
tex) for 2 min and kept in an incubator at 25 °C 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 (Iyer et al. 2006).
RESULTS AND DISCUSSION
Biodegradation mediated by indigenous micro-
bial communities is the ultimate fate of the major-
ity of oil hydrocarbon that enters the marine envi-
ronment, where hydrocarbon-degrading microor-
ganisms are ubiquitous. However, rates of
biodegradation depend on abundance and meta-
bolic ability of HDB, chemical structure of the
pollutant and environmental conditions (Ron and
Rosenberg 2014). Despite the EPEA station is not
affected by industrial activities, the increasing
maritime traffic shows the importance of moni-
toring HDB abundance and their degradation
capability in order to predict the natural deconta-
mination potential in this area.
Hydrocarbon degrading bacterial abundance
This study showed the presence of HDB at
EPEA station with abundance values ranging
between 103and 105 UFC ml-1 (Figure 2). Salini-
ty values fluctuated from 33.6 to 34.2 (Table 1)
and this variation did not seem to influence on
bacterial counts. Higher counts were associated,
in general, to warmer months (summer) (Table 1)
confirming that elevated temperatures enhanced
HDB growth as reported previously (Scheibner et
al. 2018). In addition, it is remarkable the slight
increase of HDB counts observed over time, which
could be linked to the changes in marine tempera-
ture reported within the last years (Silvestri and
Berman, 2018). These authors described, through
mathematical simulation models, the significant
temperature changes in Southwestern Atlantic
Ocean during the past years and predicted the
accelerated global warming for the next decades in
this area.
Rising temperatures directly and indirectly
impact pelagic microorganisms and aquatic food
webs leading to changes in the structure and
functioning of marine ecosystems. In this sense,
26 MARINE AND FISHERY SCIENCES 34 (1): 21-36 (2021)
warming-induced increases in bacterial activi-
ties (abundance, production and respiration)
result in higher processing of organic matter
affecting carbon flow into the microbial food
web (Scheibner et al. 2018). Even more, changes
in oceanic temperature along with pH and UVR
could disrupt key microbial-mediated services in
marine ecosystems, like bacterial pollutant
detoxification processes (Coelho et al. 2016).
Therefore, the study of microbial communities
is crucial to understand the consequences of
these continuing global and local anthropogenic
distresses on health and function of marine
ecosystems.
Finally, samples corresponding to AH0617,
AH0518 and VA1318 research cruises showed
water temperatures quite low (between 11 and
13 °C) associated to high HDB counts, suggest-
ing that other factor/s could also be impacting on
the HDB counts besides water temperature.
Carbon-source utilization
Most of the strains were able to grow on tech-
nical hydrocarbon mixtures derived from oil dis-
tillation as kerosene, gasoil and mineral oil (Table
2). These substances are usually released from
vessels either by accidental spills or deliberate
discharge (Nievas et al. 2005). On the other hand,
alkanes are major crude oil components and
despite their low water solubility various
microorganisms have the ability to utilize them as
substrate using different uptake strategies fol-
lowed by specific metabolic pathways (Guibert et
al. 2016). In general, the analyzed isolates were
able to grow on alkanes assayed as sole carbon
source, especially medium chain n-alkanes (C12-
C16) but only one strain used cyclohexane.
Polyaromatic hydrocarbons are resistant to
biodegradation because of their chemical stabili-
ty, low water solubility and high recalcitrance
27
PERESSUTTI: HYDROCARBON DEGRADING BACTERIA AT EPEA STATION
Figure 2. Abundance of hydrocarbon degrading bacteria −HDB (in duplicate) during EPEA research cruises, since 2006 to 2018.
Temperature values (°C) are indicated over the bars. Values are means ±standard deviations for two replicates.
0,0
0,5
1,0
1,5
2,0
2,5
3,0
3,5
4,0
4,5
Log HDB number (CFU ml )
-1
11
18
17 14
12 10
20
12
20
14
19
13
12
12
13
11
11
11
13 11
10
15
19
18 17
17
10 10
21
11
11
16
18
21
20
12
13 16 11
19
17
11
20 20
16
12
Month-year
Oct-2006
Nov-2006
Jan-2007
Jul-2007
Oct-2007
Oct-2008
Dec-2008
Jan-2009
Feb-2009
Mar-2009
Apr-2009
Jun-2009
Jul-2009
Aug-2009
Agu-2010
Oct-2010
Dec-2010
Jan-2011
Jun-2011
Oct-2012
Jul-2013
Aug-2013
Sept-2013
Dec-2013
Mar-2014
Feb-2015
Apr-2015
Sep-2015
Apr-2016
Sep-2016
Oct-2016
Feb-2017
May-2017
Jun-2017
Aug-2017
Sep-2017
Nov-2017
Jan-2018
Apr-2018
May-2018
Jun-2018
Jul-2018
Aug-2018
Sep-2018
Oct-2018
Dec-2018
28 MARINE AND FISHERY SCIENCES 34 (1): 21-36 (2021)
Table 2. Utilization of hydrocarbons as carbon substrate.
Strain
Hydrocarbon 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Kersosene - + + - + + + - + + + + + + + + + +
Diesel oil + + + + + + + + + + + + + + + + +
Mineral oil + - + + - + + + + + + + + + + + - -
n-Pentane - - - - - - - - - - - - - - - - - -
n-Hexane - - - - - + - - - - - - + + - - - -
n-Octane - - - - + + - + - + + - + - + - - -
n-Pentadecane + + + + + + + + + - + + + + + - + +
n-Hexadecane + + + + + + + + + + + + + + + + + +
n-Dodecane + + + + + + + + + + + - + + + - - +
Phenyldecane - - + - + + - - + - - - + - + - - -
Cyclohexane - - - - - + - - - - - - - - - - - -
Benzene - - - - - + - - - - - - - - - - - -
Toluene - - - - - - - - - - - - - - - - - -
Naphtalene + + + + + + + + + + + - + - - - - -
Anthracene - + - + + + + - - + - - + - - - - -
Phenantrene - + + + - - + + + + + + + - - - - -
19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
Kersosene + + + + + + + + + + - + + + + + - -
Diesel oil + + + + + + + + + + + + + + + + + +
Mineral oil + + + + + - + + + + + + + + + - + -
n-Pentane - - - - - - - - - - - - - - - - - -
n-Hexane + + - + - + + + + + + + + - - - - -
n-Octane + - - - - - + + + + - - - - + - + -
n-Pentadecane - - + - + + + + + + + + + - + - + +
n-Hexadecane + + + + + + + + + + + + + - + + + +
n-Dodecane + + + + + + + + + + + + + - + - - -
Phenyldecane - - + - - - + + + - + - - - - - - -
Cyclohexane - - - - - - - - - - - - - - - - - -
Benzene - - - - - - - - - - - - - - - - - -
Toluene - - - - - - - - - - - - - - - - - -
Naphtalene - + + + + - + + - - - + + + + - - +
Anthracene - - + - - - + + - + - + - - - - - -
Phenantrene - - + - - - + + + + + + + - + - - -
properties (Isaac et al. 2016). Some of the strains
in this work were found to metabolize naphatal-
ene anthracene and phenanthrene. These com-
pounds are among the 16 PAHs priority pollutants
according to the US Environmental Protection
agency (EPA), and are considered some of the
most noxious compounds in the water-soluble
fraction of oil (Abo-State et al. 2018). Finally, a
few isolates (strains 6, 13, 25 and 26) showed a
broad biodegradation profile using most of the 16
HC assayed. The carbon-source utilization exper-
iment conveys some useful information showing
the potential of the selected strains for marine
decontamination, and even some of them could
be considered as possible candidates for biotech-
nological approaches.
Isolation and characterization of phenan-
threne degrading bacteria
PAHs are among the most persistent organic
pollutants in the environment, and during the last
years there has been increasing concern about
contamination of these compounds in marine sys-
tems owing to their detrimental biological effects,
toxicity and carcinogenicity (Haritash and
Kaushik 2009). Although it is well known that
bacterial degradation plays an important role in
PAHs removal from marine environments (Dong
et al. 2015; Sakshi Sing and Haritash 2020),
nowadays most studies have reported on PAH-
degrading bacteria isolated from PAH-contami-
nated soil or sediments (Haritash and Kaushik
2009; Dell’Anno et al. 2020), and little is known
about bacteria isolated from sea or brackish
waters (Izzo et al. 2019; Govarthanan et al. 2020).
During the present study five strains named E1
to E5 were selected from enrichment cultures
with phenanthrene as sole carbon source (data not
shown). Phe is often used as a model substrate in
studies on the environmental degradation of
PAHs since its structure is found in carcinogenic
compounds as benzo[a]pyrene (Gran-Scheuch et
al. 2017).
Identification of bacterial isolates and phyloge-
netic analysis
Sequence analysis of the 16S rRNA gene of the
isolates E1, E2 and E3 allowed to determine their
relationship to the genus Halomonas (Figure 3).
E1 strain was closely related to Halomonas sp.
XJ10 (99.19 %) and also with Salinicola sp. ATA
24 (99.35%). Similarly, E2 strain was associated
to Halomonas sp. CR-55 (99.26%) and H. merid-
iana RT31 (99,14%), while E3 strain was related
to H. nanhaiensis MTA-40-2-2 and H. sulfidaeris
M-143 with a similarity value of 99.87%.
Halomonas are slight to moderately halophilic
and oligotrophic organisms that are ubiquitous to
marine and hypersaline environments, and grow
under different environmental conditions.
Although among halophilic bacteria, Halomonas
sp. has been shown to utilize a wide range of
readily available substrates as energy sources for
its fast growth (Ali et al. 2016) and is multi-metal
resistant (Govarthanan et al. 2017), to our knowl-
edge there are few studies on PAH degrading
Halomonas isolated from marine waters (Gasper-
otti et al. 2015; Corti Monzón et al. 2018; Izzo et
al. 2019; Govarthanan et al. 2020). It is worth
noting that H. meridiana and H. sulfidaeris
strains retrieved from deep sea sediments were
also previously reported as PAH degraders (Cui et
al. 2008; Yuan et al. 2015).
On the other hand, strain E4 showed closest
association with Rhodococcus sp. Voy40th18-6
and R. erythropolis MC15 (97.78%). Bacteria
belonging to Rhodococcus sp. have been charac-
terized by their enormous metabolic versatility
and by the concomitant metabolic bioconversion
reactions of structurally diverse HC in marine
systems (Brzeszcz and Kaszycki 2018). Like-
wise, R. erythropolis and other Rhodococcus
members are able to degrade PAHs through dif-
ferent catabolic pathways (Seo et al. 2009).
Finally, strain E5 belongs to the genus
Pseudomonas since its 16S rDNA sequence was
closely related to those of Pseudomonas rhodesi-
ae 15D2 (98.20%) and Pseudomonas sp. SI4
29
PERESSUTTI: HYDROCARBON DEGRADING BACTERIA AT EPEA STATION
(98.20%). Pseudomonas sp. is one of the most
studied genus and has been reported as a degrader
of a wide range of organic pollutants including
PAHs and other recalcitrant xenobiotics (Mulet et
al. 2011). PAH degraders from coastal marine
environments have been also described as bacte-
ria associated to this genus (Isaac et al. 2016).
Particularly, P. rhodesiae has shown ability to
grow rapidly in PAHs (Kahng et al. 2002).
Phenanthrene biodegradation
The ability to estimate PAH degradation rates
is essential for predicting environmental fate and
for designing remediation efforts (Mallick and
Dutta 2008). Biodegradation of phenanthrene
used as the sole carbon and energy source was
analyzed by HPLC for five isolates at an initial
concentration of 160 mg l-1 (Figure 4). A contin-
uous degradation curve from the initiation of the
assay was observed for Halomonas sp. strains
E1, E2 and E3, reaching a substrate disappear-
ance between 37 to 45%. Phenanthrene concen-
tration decreased more rapidly in the first four
days for strain E1 and in the first six days for
strains E2 and E3 than in later days, remaining
steady after ten days of incubation. This might
be attributed to the higher concentration of sub-
strate at the beginning and the inhibited degrada-
tion of phenanthrene to some extent by metabo-
lites later.
For Rhodococcus sp. strain E4 and Pseudo-
monas sp. strain E5, instead, a fast drop in Phe
concentration was detected during the first 48 h,
continuing with a slower decrease as the experi-
ment progressed. The initial drop could be due to
an active cellular assimilation or adsorption to the
cell wall as was observed by Tian et al. (2002),
who proposed that real degradation would occur,
at least partially, after the early assimilation or
adsorption and it should be considered during the
discussion of degradation kinetics. This highly
insoluble hydrocarbon was completely used by
30 MARINE AND FISHERY SCIENCES 34 (1): 21-36 (2021)
Figure 3. NJ phylogenetic tree based on an approximately 1,300 bp segment of the 16S rRNA gene sequence of PAHs degrading
strains from this work and related sequences. GenBank accession numbers are given in parentheses. Only bootstrap val-
ues higher than 50% out of 1,000 replications are shown. Bar represents 0.05 nucleotide substitutions per site.
Halomonas nanhaiensis MTA-40-2-2 (KJ401091)
Halomonas sulfidaeris strain M-143 (KF177272)
Halomonas meridiana strain RT31 (KC842226)
Halomonas sp. CR-55 (KT324894)
Salinicola sp. ATA 24 (LC385628)
Halomonas sp. strain XJ10 (MN715851)
Pseudomonas rhodesiae 15D2 (MG269606)
Pseudomonas sp. SI4 (JF519724)
Rhodococcus sp. Voy40th18-6 (MT588453)
Rhodococcus erythropolis MC15 (LT984719)
E3
E2
E1
E5
E4
90
68
79
100
88
99
96
100
99
100
97
74
0.050
strains E4 and E5 after 6 and 10 incubation days,
respectively. By contrast, the biodegradation
level of most bacteria reported in the literature
was found to be below 50%, even after 10-day
incubation (Song et al. 2011; Thavamani et al.
2012). Degradation rates were as follow: strains
E1, E2 and E3: 4.92, 6.0 and 5.58 mg l-1 day-1,
respectively; whereas strains E4 and E5: 26.66
and 16.0 mg l-1 day-1, respectively. These values
were concomitant with OD measurements from
the strain cultures (data not shown). Unlike other
studies (Yuan et al. 2000) the addition of simpler
carbons sources, as glucose, was not necessary to
enhance PAH biodegradation rates.
Finally, degradations by Rhodococcus sp. and
Pseudomonas sp. strains were around 2-3 times
higher compared to Halomonas sp., indicating
that potentials inherent in a genus and its species
are also crucial considerations in the biodegrada-
tion of these aromatic compounds. Nevertheless,
exhaustive information on the biodegradation of
PAHs in seawaters by halophilic/halotolerant
bacteria is still an emerging field in its initial
stage of exploration (Ghosal et al. 2016).
Biosurfactant production assays
Many PAH-degrading bacteria have developed
different strategies to overcome the low aqueous
solubility of hydrocarbons (HC). One of the main
HC accession processes is the production of sur-
face-active agents (Olivera et al. 2009). Biosur-
factants are amphipathic molecules secreted to
the environment, which enhance solubilization
and elimination of contaminants. Their action
mechanism lies in the accumulation of immisci-
ble compound at the interface, diminishing the
surface tension and thus increasing their surface
area; this allows a higher bioavailability that
facilitates the degradation of diverse pollutants as
aromatics (Batista et al. 2006). Microorganisms
able to increase the degradation of hydrophobic
compounds by releasing biosurfactants usually
belong to the Genera Pseudomonas, Halomonas,
Bacillus, Rhodococcus and Stenotrophomonas
(Tripathi et al. 2020). In this study, biosurfactant
production was determined by droplet collapsing
test and hexadecane emulsification abilities of
cell-free culture media. Supernatant from cultures
of the five analyzed strains indicated the presence
31
PERESSUTTI: HYDROCARBON DEGRADING BACTERIA AT EPEA STATION
0
20
40
60
80
100
120
140
160
180
0246810
12
E1 E2 E3 E4 E5
Time (days)
Phenanthrene (mg l )
-1
Figure 4. Degradation of phenanthrene by isolated strains.
of surface-active compounds in both assays
(Table 3), showing relatively high emulsifying
indexes and positive drop collapsing activity in
strains E1, E2, E3 and E4. The significant biosur-
factant activity observed in Halomonas and
Rhodococcus strains suggested somehow that the
surfactant secreted to the medium would be
involved in Phe degradation.
Various Halomonas species have been reported
to produce abundant quantities of surface-active
agents, as exopolymeric substances (EPS), which
may provide a tool to scavenge hardly soluble,
hydrophobic substrates, that cells could then uti-
lize for growth in marine environments (Gutier-
rez et al. 2020). In addition, previous reports
showed that several members of Rhodococcus
produce biosurfactants, and even some species as
R. eritropolis are regarded as natural reservoirs of
new biosurfactants (Peng et al. 2007).
CONCLUSIONS
In this study, knowledge about the intrinsic HC
biodegradation potential by native microbial com-
munities around the EPEA station (Atlantic Coast)
was first revealed. An increasing bacterial abun-
dance associated with temperature over time was
detected, indicating that further studies are needed
to evaluate how climate change, anthropogenic
pollution, and microbiological interactions may
affect marine ecosystems in the future. In addi-
tion, some HDB isolated during this research were
able to utilize technical hydrocarbon mixtures,
alkanes, cycloalkanes and/or polyaromatic hydro-
carbons (PAHs) as substrate, showing their poten-
tial for marine decontamination.
Finally, the five Phe-degrading bacteria select-
ed and characterized in this study exhibited phys-
iological properties that might have ecological
significance on environmental alterations as the
presence of pollutants. It is worth noting that
Rhodococcus sp. strain E4 showed an outstanding
PHA degrading capacity and significant biosur-
factant activity. This strain could be exploited for
biotechnological applications, as the develop-
ment of cost-effective and eco-friendly technolo-
gies for the removal of PAHs in diverse marine
environments.
ACKNOWLEDGEMENTS
This research was supported by the Instituto
Nacional de Investigación y Desarrollo Pesquero
(INIDEP). The author thanks the valuable collab-
oration of Constanza Hozbor who collected the
samples during the research cruises, and also
gratefully acknowledge the contribution of Gra-
ciela Molinari for providing oceanographic data.
INIDEP contribution no 2233.
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