APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 2009, p. 3407–3418
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 75, No. 11
Bacterial Communities from Shoreline Environments (Costa da Morte,
Northwestern Spain) Affected by the Prestige Oil Spill?†
Jorge Alonso-Gutie ´rrez,1Antonio Figueras,1Joan Albaige ´s,3Nu ´ria Jime ´nez,2,3Marc Vin ˜as,2,4
Anna M. Solanas,2and Beatriz Novoa1*
Instituto de Investigaciones Marinas, CSIC, Eduardo Cabello 6, E-36208 Vigo, Spain1; Department of Microbiology, University of
Barcelona, Diagonal 645, E-08028 Barcelona, Spain2; Department of Environmental Chemistry, IIQAB-CSIC, Jordi Girona 18-26,
08034 Barcelona, Spain3; and GIRO Technological Center, Rambla Pompeu Fabra 1, E-08100 Mollet del Valle `s, Spain4
Received 1 August 2008/Accepted 26 March 2009
The bacterial communities in two different shoreline matrices, rocks and sand, from the Costa da Morte,
northwestern Spain, were investigated 12 months after being affected by the Prestige oil spill. Culture-based and
culture-independent approaches were used to compare the bacterial diversity present in these environments
with that at a nonoiled site. A long-term effect of fuel on the microbial communities in the oiled sand and rock
was suggested by the higher proportion of alkane and polyaromatic hydrocarbon (PAH) degraders and the
differences in denaturing gradient gel electrophoresis patterns compared with those of the reference site.
Members of the classes Alphaproteobacteria and Actinobacteria were the prevailing groups of bacteria detected
in both matrices, although the sand bacterial community exhibited higher species richness than the rock
bacterial community did. Culture-dependent and -independent approaches suggested that the genus Rhodo-
coccus could play a key role in the in situ degradation of the alkane fraction of the Prestige fuel together with
other members of the suborder Corynebacterineae. Moreover, other members of this suborder, such as Myco-
bacterium spp., together with Sphingomonadaceae bacteria (mainly Lutibacterium anuloederans), were related as
well to the degradation of the aromatic fraction of the Prestige fuel. The multiapproach methodology applied
in the present study allowed us to assess the complexity of autochthonous microbial communities related to the
degradation of heavy fuel from the Prestige and to isolate some of their components for a further physiological
study. Since several Corynebacterineae members related to the degradation of alkanes and PAHs were fre-
quently detected in this and other supralittoral environments affected by the Prestige oil spill along the
northwestern Spanish coast, the addition of mycolic acids to bioremediation amendments is proposed to favor
the presence of these degraders in long-term fuel pollution-affected areas with similar characteristics.
Since the Polycommander accident, many other oil spills,
such as the Urquiola (1976), Andros Patria (1978), and Aegean
Sea (1992) spills, have occurred on the Galician coast (north-
western Spain), where intense maritime traffic takes place. On
13 November 2002, the oil tanker Prestige sprang a leak off
Cape Finisterre (Galicia, northwestern Spain) and 6 days later
its oil tank broke up and sank 240 km west of Galicia. The spill
of 60,000 tons of heavy fuel oil polluted 500 miles of the
Spanish coast, reaching the French coast. The Costa da Morte,
northwestern Spain, was the most affected area (2). The oil
residue released by the Prestige was devoid of the more labile
fractions (boiling point, ?300°C), with high levels of aromatic
hydrocarbons (?50%), as well as resins and asphaltenes
Information about the autochthonous microbial populations
at maritime oil-polluted sites is scarce (23). The studies carried
out after the Nakhodka spilled oil with a chemical composition
similar to that of the Prestige fuel, gathered information on the
marine microbial populations that adapted to heavy fuel oil.
Different molecular approaches, mainly involving 16S rRNA
gene analyses such as PCR-denaturing gradient gel electro-
phoresis (DGGE) (31), clone libraries, and specific oligonu-
cleotide probes (43), were used to describe the bacterial com-
munities established in different environments and at different
time intervals after the oil spill.
Most of the previous studies were focused either on the
isolation of a few culturable degrading strains or just on de-
tecting the 16S rRNA gene sequences of all of the bacteria
present in polluted samples without gathering information on
their physiology. As a consequence, more efforts should be
made to understand community structures in situ and to isolate
the key oil-degrading species present, with the aim to further
investigate their requirements (23, 58) that could be used in
the development of new bioremediation.
In the present report, we describe a microbiological analysis
of a cobblestone beach on the Costa da Morte, northwestern
Spain, affected by the Prestige heavy fuel oil spill 12 months
after the last fuel stranding. The microbial community was
examined thoroughly by a triple-approach method based on
different cultivation strategies and culture-independent meth-
ods such as DGGE and the screening of 16S rRNA gene clone
MATERIALS AND METHODS
Sampling. In March 2004, 12 months after the last fresh fuel stranding from
the Prestige (Fig. 1), oil-polluted samples were taken from the supralittoral zone
of a cobblestone beach located next to Faro Larin ˜o (42°46?25?N, 09°07?30?W,
* Corresponding author. Mailing address: Instituto de Investigacio-
nes Marinas (CSIC), Eduardo Cabello 6, E-36208 Vigo, Spain. Phone:
(34) 986 214463. Fax: (34) 986 292762. E-mail: email@example.com.
† Supplemental material for this article may be found at http://aem
?Published ahead of print on 17 April 2009.
at CSIC-PUBLICACIONES ELECTRONICAS on June 11, 2010
Carnota, Spain). Samples included small oil drops scattered among sand grains
(OS) and fuel paste attached to rock surfaces (OR). The heavy fuel from the
Prestige attached to rock surfaces and interstices formed thick oil layers where
different materials get attached. Nonoiled sand (NOS) samples from an adjacent
zone were taken as controls. Samples were placed in sterilized glass jars and kept
cool (4°C) or frozen (?20°C) until analysis (Fig. 2).
Chemical analysis. To assess the degree of biodegradation of the sample and
to verify that no cross contamination from sources other than the Prestige fuel
occurred, oil residues (1 g) were dissolved in 5.0 ml of dichloromethane (Supra-
Solv grade; Merck, Darmstadt, Germany), phase separated, and percolated
through 2 g of anhydrous sodium sulfate. The organic extracts were carefully
evaporated until dried, and one aliquot (5 to 10 mg) was dissolved in hexane and
then fractionated in a previously conditioned (with 6 ml hexane; Merck) cyano-
propyl silica solid-phase cartridge (SiO2/CN, 1.0/0.5 g, 6 ml; Interchim, Montlu-
c ¸on, France) as reported elsewhere (6). The aliphatic and aromatic fractions
were obtained by elution with 4.0 ml of hexane (fraction 1) and 5.0 ml of
hexane-dichloromethane (1:1) (fraction 2), respectively. Both fractions were
then analyzed by gas chromatography-mass spectrometry on a TRACE-MS
Thermo Finningan TRACE-GC 2000 gas chromatograph (Thermo Finningan;
Dreieich, Germany) fitted with an HP 5MS (30 m by 0.25 mm [inside diameter]
by 0.25 ?m film) capillary column (J&W Scientific, Folsom, CA).
The extent of biodegradation of each compound was measured from the
normalized peak area of the target analyte referred to that obtained from the
same compound in the control sample (2, 28). The peak areas of the target
analytes were measured in the reconstructed ion chromatograms at m/z 85 for
aliphatics and at the corresponding molecular ion for the aromatics as described
Microbial characterization. Samples were analyzed directly (OR, OS) or en-
riched first with different fuel components such as alkanes or aromatics. The
procedures and nomenclature used are summarized in Fig. 2 and Table 1,
Enumeration of heterotrophic and hydrocarbon-degrading microbial popula-
tions. Bacterial counts of heterotrophs and n-hexadecane and polyaromatic
hydrocarbon (PAH) degraders were performed by a miniaturized most-probable-
number (MPN) method in 96-well microtiter plates with eight replicate wells per
dilution as described elsewhere (3, 69). All of the media used (tryptic soy broth
and mineral medium BMTM ) were corrected to reach 3% NaCl. MPN
analysis results are shown as means of triplicates, and the Student t test was used
to compare them (Fig. 3).
Isolation of culturable strains. Culturable microorganisms from OR (rock)
and OS (sand) samples and from enrichment cultures grown on phenanthrene or
n-hexadecane were isolated onto different media (Table 1). Culturable hetero-
trophs were isolated at 20°C onto fivefold-diluted marine agar (MA 1/5) supple-
mented to maintain 3% NaCl. n-Hexadecane and phenanthrene degraders were
isolated on mineral agar (BMTM agar, 3% NaCl) supplemented with n-hexade-
cane in the vapor phase (54) or phenanthrene (0.1%) as a sole carbon and energy
source, respectively (Table 1). All isolated strains were stored at ?80°C in 20%
(vol/vol) glycerol for subsequent analysis.
Screening of the hydrocarbon-degrading capability of strains. All of the iso-
lated strains were screened for the ability to degrade alkanes and aromatics on
either solid or liquid mineral medium as previously described (3).
FIG. 2. Flow chart diagram illustrating the protocols used in this
study. For both sample types (OR and OS), chemical, microbiological,
and molecular analyses were done. Templates for PCR-DGGE were
DNA extracted directly from the environment (samples A/DNAs a, in
boxes), from the trophic populations (pop.) grown in MPN analysis
plates (samples B and C/DNAs b and c, in boxes), or from degrading
strains. The DGGE profiles of the total DNA of each sample and of its
trophic populations (heterotrophic and alkane and aromatic degrad-
ing) were compared to identify common bands between them. Degrad-
ing strains isolated with different media were individually screened by
DGGE to detect those that comigrated with specific bands in the
profiles. A nearly full-length 16S rRNA gene PCR fragment from the
total DNA (a) of the samples was cloned. The clone library was
screened by restriction fragment length polymorphism analysis, and
different operational taxonomic units were sequenced. Sequences from
the most interesting bands and strains were compared with those from
the clone library to determine the quantitative proportions of the
different species found.
FIG. 1. (A) Detailed map of the northwestern coast of Spain (Galicia) known as the Costa da Morte or the Costa de la Muerte (from the Muros
and Noia Estuary to Malpica), which means Coast of Death, for its strong swell and harsh weather. Punta I´nsua is the main landmark next to the
sampling site. (B) Closer image of the sampling site.
3408 ALONSO-GUTIE´RREZ ET AL.APPL. ENVIRON. MICROBIOL.
at CSIC-PUBLICACIONES ELECTRONICAS on June 11, 2010
To assess hydrocarbon-degrading capability in solid medium, mineral agar
supplemented with n-hexadecane and phenanthrene was used as described
above. Microtiter plates containing 200 ?l per well mineral medium (BMTM, 3%
NaCl) and n-hexadecane, F1, or a PAH mixture were used, as for MPN analysis,
in liquid screenings. F1 is the aliphatic fraction (2.5 g ? liter?1) obtained from
Casablanca crude oil (61).
To inoculate the biodegradation assays, the strains were grown overnight at
room temperature on tryptic soy broth (3% NaCl). Cells were harvested by
centrifugation at 4,000 ? g for 15 min, washed twice, and finally suspended in
mineral medium (BMTM plus 3% NaCl) to reach an optical density of 0.5
(determined at 620 nm with a Multiskan spectrophotometer [Labsystems]).
Twenty microliters of suspended cells was used for the inoculation of two wells
per plate. Another plate with only mineral medium was inoculated as a negative
control. Only those wells with evident turbidity compared to the control plate
were considered positive.
DNA extraction. Total community DNA was extracted from OR and OS
samples by a bead-beating protocol with a PowerSoil DNA soil extraction kit
(MoBio Laboratories, Inc., Solano Beach, CA) by following the manufacturer’s
Genomic DNA from the heterotrophic population and from those related to
alkane and aromatic compound degradation (Hx and PAHs, respectively) was
obtained from the eight wells corresponding to the highest positive dilution of
plates of OR and OS samples subjected to MPN analysis. Cells were harvested
from wells, lysed with sodium dodecyl sulfate (10%), lysozyme, and proteinase K;
treated with 10% cetyltrimethylammonium bromide; and freeze-thawed three
times with liquid nitrogen and a 65°C bath. The extracted DNA was purified by
phenol-chloroform-isoamyl alcohol extraction as previously described (10, 67).
DGGE. Genomic DNA from OR, OS, NOS, MPN analysis microtiter plates,
and hydrocarbon-degrading strains were subjected to DGGE analysis. 16S rRNA
gene hypervariable regions V3 to V5 were amplified with primers 16F341-GC and
16R907 (73). Primer F341-GC included a GC clamp at the 5? end (5?-CGCCCGC
CGCGCCCCGCGCCCGTCCCGCCGCCCCCGCCCG-3?). In this case, PCRs
were performed in a volume of 50 ?l containing 1.25 U of Taq (TaKaRa ExTaq
Hot Start Version; TaKaRa Bio Inc., Otsu, Shiga, Japan), 1? ExTaq buffer (2
mM MgCl2), 200 ?M each deoxynucleoside triphosphate, 0.5 ?M primers, and
100 ng of template DNA. After 9 min of initial denaturation at 95°C, a touch-
down thermal profile protocol was carried out and the annealing temperature
was decreased by 1°C per cycle from 65 to 55°C, followed by 20 additional cycles
of 1 min of denaturation at 94°C, 1 min of primer annealing at 55°C, and 1.5 min
of primer extension at 72°C and then a final 10 min of primer extension at 72°C.
Approximately 800 ng of purified PCR product was loaded onto a 6% (wt/vol)
polyacrylamide gel that was 0.75 mm thick with denaturing gradients and dena-
turant concentrations that ranged from 40 to 75% (100% denaturant contained
7 M urea and 40% formamide). DGGE was performed in 1? TAE buffer (40
mM Tris, 20 mM sodium acetate, 1 mM EDTA, pH 8.4) with a DGGE-2001
system (CBS Scientific Company, Del Mar, CA) at 100 V and 60°C for 16 h.
DGGE gels were stained with 1? TAE buffer containing Sybr gold (Molecular
Probes, Inc., Eugene, OR). Predominant DGGE bands were excised with a
sterile razor blade, suspended in 50 ?l sterilized MilliQ water, stored at 4°C
overnight, reamplified by PCR with primers F341 and R907, and cloned with a
TOPO TA cloning kit (Invitrogen) as described below.
Analysis of DGGE images. Bacterial diversity analysis and correlation princi-
pal-component analysis (PCA) of band types were performed, and the relative
peak areas were calculated as previously described (62) for the different DGGE
profiles (i.e., OR, OS, NOS, OR-Hx, OR-PAH, OS-Hx and OS-PAH [Fig. 4]) to
consider possible shifts in the composition of the microbial populations. A
dendrogram was constructed by the nearest-neighbor cluster method with the
TABLE 1. Nomenclature of sequences retrieved in this study sorted by their methodological sources and origins as shown in the flow chart
diagram in Fig. 1a
Sample and source (code)
OR samplesOS samples
Figure(s) Sequence code
DGGE bands from PCR-amplified:
Total DNA (a)
DNA from MPN Hx (b)
DNA from MPN PAHs (c)
R ? ner
RH ? ner
RPb ? ner
S ? ner
SH ? ner
SP ? ner
Clone library from PCR-amplified
total DNA (a)
Rc ? ner
S4 Sc ? ner
Strains isolated with:
MA 1/5 from original samples (A)
MA 1/5 from highest dilution in
Hx MPN analysis (B)
MA 1/5 from highest dilutions in
PAH MPN analysis (C)
BMTM ? Hx from original
BMTM ? Phe from original
RP ? ner
RPH ? ner
AP ? ner
APH ? ner
RPP ? ner
S1, S5APP ? ner
PDR ? ner
S1, S5 6A PDA ? ner
S1, S6 6B
PhR ? ner
S1, S56C PhS ? ner
S1, S6 6C
aFigures and tables in the supplemental material related to Fig. 1 samples are indicated for clarity. MA, marine agar; BMTM, mineral agar; Phe, phenanthrene; Hx,
n-hexadecane; Hx MPN or PAH MPN, plate counting of MPN of microbial populations related to alkane or aromatic degradation, respectively. A, B, and C, sample
codes indicated in the flow chart diagram in Fig. 2. a, b, and c, DNA extracted from A, B, and C samples, respectively, as shown in Fig. 1.
FIG. 3. MPN analysis of heterotrophic (HET), alkane-degrading
(ALK), and PAH-degrading populations (PAHs) in polluted samples
of rocks (OR) and sand (OS) compared with those in nonoiled sand
(NOS). Standard deviations (n ? 8) are represented by error bars.*,
significantly different from the other two samples by the Student t test.
Polluted samples have similar populations, except for aromatic degrad-
ers, while unpolluted samples always have significantly smaller degrad-
ing populations than polluted ones do.
VOL. 75, 2009 PRESTIGE OIL-POLLUTED SHORELINE BACTERIAL COMMUNITIES3409
at CSIC-PUBLICACIONES ELECTRONICAS on June 11, 2010
Pearson product-moment correlation coefficients calculated from the complete
densitometric curves for the fingerprints of the different bacterial communities.
16S rRNA gene clone library. Almost the complete 16S rRNA gene was
amplified from OS and OR genomic DNA with primers F27 and R1492 as
previously described (19, 35). The PCR mixture (25 ?l) included 10 mM Tris HCl
(pH 8.3), 50 mM KCl (pH 8.3), 2.5 mM MgCl2, 200 ?M each deoxynucleoside
triphosphate, 1.25 U of AmpliTaq Gold DNA polymerase (PE Applied Biosys-
tems, Foster City, CA), 0.4 ?M each primer, and 100 ng of DNA extracted from
either OR or OS samples. The reaction mixtures were subjected to an initial
denaturation and enzyme activation step (5 min at 95°C); 40 cycles of 30 s at
96°C, 30 s at 54°C, and 1.5 min at 72°C; and an extension step of 10 min at 72°C.
PCR products were ligated into the pCR2.1-TOPO vector and transformed
into competent Escherichia coli TOP 10F? cells by following the protocol of the
manufacturer of the TOPO T/A cloning kit (Invitrogen). Restriction fragment
length polymorphism analysis of the clones was performed to identify clone
representatives of different enzyme restriction patterns, digesting the PCR prod-
ucts separately with 5 U of AluI and TaqI (Amersham Biosciences, Uppsala,
Sweden) for 3 h at 37°C and 65°C, respectively.
PCR products from recombinant clones and the resulting restriction enzyme
fragment patterns were separated by electrophoresis in a 1% and 3% (wt/vol)
agarose gel in 1? TAE buffer, respectively, stained with ethidium bromide, and
photographed under UV light with a Gel Doc XR system and Quantity One
software (Bio-Rad, Hercules, CA). Clones representatives of different enzyme
restriction patterns were sequenced in both directions with internal primers F341
and R907 (19).
Sequencing and phylogenetic analysis. Sequencing was accomplished with the
ABI PRISM Big Dye terminator cycle sequencing ready reaction kit (version 3.1)
and an ABI PRISM 3700 automated sequencer (PE Applied Biosystems, Foster
City, CA) by following the manufacturer’s instructions. 16S rRNA genes were
partially sequenced in both directions with primers F341 and R907 (19). Se-
quences were inspected, assembled, subjected to the Check Chimera program of
the Ribosomal Database Project (41), and examined with the BLAST search
alignment tool comparison software (BLASTN) (4) to detect the bacterial group
in the GenBank database closest to each strain.
Sequences were aligned with reference sequences obtained from GenBank,
and phylogenetic analyses were performed as previously described (3) to better
classify the detected bacteria.
Nucleotide sequence accession numbers. The 363 nucleotide sequences iden-
tified in this study have been deposited in the GenBank database under accession
numbers EU374875 to EU375237.
Chemical analysis. The gas chromatographic profiles of the
aliphatic fractions evidenced petrogenic contamination based
on the occurrence of the homologous series of C15to C40
n-alkanes overlying an unresolved complex mixture of hydro-
carbons (see Fig. S1 in the supplemental material). The con-
firmation of the presence of the Prestige oil was obtained by a
detailed study of the fossil biomarkers, namely, steranes and
triterpanes, currently used for oil spill fingerprinting (15). The
diagnostic molecular parameters of the oiled samples indicated
a clear correspondence to those of the fuel oil (see Fig. S2 in
the supplemental material), whereas those of the control sand
sample (NOS) exhibited a different pattern. The ratios of C2
and C3dibenzothiophenes (D2 and D3) and phenanthrene/
anthracenes (P2 and P3), proposed for differentiating sources
of spilled oils in sediments (17), also supported the presence of
the Prestige oil in the collected samples (see Fig. S2 in the
The occurrence of biodegradation was assessed by the de-
pletion of certain components with respect to those more re-
fractory, such as triterpanes (e.g., hopane), and by changes in
relative distributions within isomeric series (e.g., alkyl C1- and
C2-phenanthrenes, dibenzothiophenes). In summary, the n-
alkanes were severely depleted in the lower fraction (?n-C20)
as a result of weathering, but the ones in the higher fraction
were also, which should be attributed to biodegradation (see
Fig. S3A in the supplemental material).
Enumeration of heterotrophic, alkane-degrading, and aro-
matic-degrading microbial populations. While the total het-
erotrophic bacteria in the oiled and nonoiled sands (OS and
NOS) presented similar abundances (105to 106microorgan-
isms per g of sample), hydrocarbon-degrading populations
were 10- to 100-fold greater in the oiled sample than in NOS
(Fig. 3). The alkane-related populations found in OR and
FIG. 4. DGGE profiles of PCR-amplified 16S rRNA genes of bacterial communities from oiled samples (OR and OS) compared with those
of bacterial communities from NOS (A). The hydrocarbon (alkane [Hx] and aromatic compound [PAH])-degrading bacterial populations from
polluted rock (B) and sand (C) are compared with their total profiles (OR, OS). R1 to R18 and S1 to S13 indicate excised and sequenced bands
from the total OR and OS profiles, respectively (see Table S2 in the supplemental material). Bands of the alkane (OR-Hx and OS-Hx)- and
aromatic (ORPx and OSPx)-degrading populations from rock and sand, respectively, were sequenced as well (see Table S3 in the supplemental
material). Bacterial diversity (Table 3), PCA (see Fig. S4 in the supplemental material), and a neighbor-joining tree from Pearson correlation factor
data (see Fig. 5) were obtained from the complete densitometric curves of the different DGGE profiles (i.e., OR, OS, NOS, OR-Hx, OR-PAH,
OS-Hx, and OS-PAH) to consider possible shifts in the composition of the microbial populations.
3410 ALONSO-GUTIE´RREZ ET AL.APPL. ENVIRON. MICROBIOL.
at CSIC-PUBLICACIONES ELECTRONICAS on June 11, 2010
OS were also similar (around 104to 105microorganisms ?
g?1), accounting for more than 50% of the heterotrophic
bacteria and always greater than the aromatic one. How-
ever, aromatic-degrading bacterial counts were 10-fold
higher in the polluted sand (103?104microorganisms ?
g?1) than in the oiled rocks.
Isolation of culturable strains. Around 40 morphologically
different strains were isolated on MA 1/5 directly from each
sample (RPx and APx strains), whereas more than 20 strains
were isolated in the same medium from the MPN analysis
plates of populations related to n-hexadecane and aromatic
hydrocarbon degradation (RPHx and RPPx strains from OR
and APHx, APPx strains from OS; Table 1).
With selective medium for isolation of alkane degraders
(BMTM agar plus n-hexadecane), approximately 20 additional
strains were isolated directly from rock (PDRx strains) and
sand (PDAx strains) samples, respectively. Finally, 15 addi-
tional strains from each sample were isolated in phenanthrene
agar from an enrichment culture grown on phenanthrene
(0.05%, wt/vol) at 150 rpm and 25°C for more than 2 weeks
(strains PhRx and PhSx, Table 1).
All of the strains isolated from polluted sites (RP and AP)
were sequenced, but in the other cases (RPH, RPP, APH,
APP, PDA, PDR, PhR, and PhS) only those strains suspected
of having some degrading capacity or with the same migration
length as any of the OR/OS-related DGGE bands were ana-
lyzed further (Table 1).
Screening of hydrocarbon-degrading capability. Alkane-de-
grading activity was found in isolated strains from both of the
environments studied (sand and rock), and although the per-
centage of degraders varied depending on the medium used, it
was always dominated by Actinobacteria (see Table S1 in the
supplemental material). In general, polluted sand samples (see
Table S6 in the supplemental material) presented a much
higher percentage of hydrocarbon-degrading strains than did
polluted rock samples (see Table S5 in the supplemental ma-
terial), even with nonselective medium (33% of AP strains
compared to 2.6% of RP strains). As expected, the use of a
selective medium (hydrocarbon-agar) was the best strategy to
isolate alkane-degrading strains (72 and 81% of the PDR and
PDA strains were alkane degraders; see Table S1 in the sup-
plemental material) and almost the only way to isolate bacteria
related to PAH degradation (PhR/PhS strains). Eleven PhR
and nine PhS isolates (representing 73 and 60% of the total)
from the OS and OR phenanthrene enrichments grew as pure
cultures on PAHs. These strains belonged to only two species
of Pseudomonas and Sphingomonas (Table 2).
DGGE profiles of the total bacterial community. Cluster
analysis and PCA indicated that the bacterial communities
from oiled samples (OR and OS) were quite similar, while the
nonoiled control (NOS) was the most distantly related (Fig. 5;
see Fig. S4 in the supplemental material). Five OR DGGE
bands, R1, R2, R7, R14, and R15, were identical in sequence
to OS DGGE bands S1, S2, S7, S10, and S11, respectively.
Those bands were related to the genus Rhodococcus, uncul-
tured Rhodobacteraceae, Lutibacterium, and Chromatiales, re-
spectively. Additional bands from these and other organisms
related to oil degradation, such as Citreicella spp. (bands R9,
R10, and R11), Sphingopyxis spp. (bands R12 and R17), Eryth-
robacter spp. (band R16), and Yeosuana aromativorans (band
S13), were also found in total DGGE profiles (Table 2 see
Table S2 in the supplemental material).
DGGE profiles of presumably oil-degrading bacterial pop-
ulations. PCA (see Fig. S4 in the supplemental material) of
excised and nonexcised bands from the different DGGE pro-
files (Fig. 4B and C) suggested that OR community members
were mainly related to alkane degradation while the oiled sand
community included members related to the degradation of
both fractions. However, 16S rRNA gene sequences were ob-
tained only from the most conspicuous bands of the profiles
and no sequences common to populations related to the deg-
radation of alkanes (Hx) and aromatics (PAHs) (see Table S3
in the supplemental material) and the total community (OR,
OS) could be confirmed (see Table S2 in the supplemental
material). An exception was found with bands RH1 and RH2
from the presumably n-hexadecane-degrading population pro-
file of the rock sample (OR-Hx). These bands were, respec-
tively, identical to the Rhodococcus bands shared by the total-
community profiles of the oiled rock and sand (RH1 ? R1 ?
S1 and RH2 ? R2 ? S2; Table 2).
Higher diversity related to alkane and aromatic degradation
was found in the sand than in the oiled rocks (Table 3). How-
ever, some common bands within the OR-Hx profiles (RH4)
and OS-Hx (SH5, SH6) were found related to Pseudoxan-
thomonas spadix (99 to 100% similarity) (Table 2). Other Xan-
thomonadaceae genera related to alkane degradation in OS
were close to Dokdonella koreensis (bands HA2/HA3) and to
Stenotrophomonas maltophilia (band SH8). The Alphapro-
teobacteria genus Erythrobacter was detected as one of the most
conspicuous of the OS-Hx profile (SH7).
The OR-PAH DGGE profile was composed of only two
dominant bands (Fig. 4B), RPb1 and RPb2, corresponding to
the genera Tistrella and Sphingomonas, whereas eight bands
could be excised and sequenced from the OS-PAH DGGE
profile (Fig. 4C; see Table S3 in the supplemental material). It
is important to point out that band RPb2 (OR-PAHs) was
identical to SP7 (OS-PAHs), being close to Sphingomonas spp.
Another three similar sequences from sand, bands PA5,
PA6, and PA8, were also related to the genus Sphingomonas
Clone libraries. As explained in the following section, to
obtain an image of the most abundant genera present in each
matrix community (Table 2; see Table S4 in the supplemental
material), approximately 70 clones were sequenced for each
sample (OR and OS).
Oiled rock (OR) sample total community. The main bacte-
rial groups found in OR were the classes Alphaproteobacteria
(43%; the genera Parvibaculum and Lutibacterium), Actinobac-
teria (28%; Rhodococcus, Dietzia, and Microbacterium spp.),
and Gammaproteobacteria (23%; Salinisphaera, Chromatiales,
and Alcanivorax spp.) (see Table S1 in the supplemental ma-
terial). The most important genus was Rhodococcus, which was
represented by seven different sequences accounting for 20%
of the total library. Two of these sequences, with the highest
frequencies, were identical to DGGE bands R1 and R2, re-
spectively (Table 2). Phylogenetic analysis placed them close to
Rhodococcus fascians DSM20669 (99 and 98% similarity, re-
spectively). A minor presence of clones related to Alcanivorax
spp. (3 out of 65) was found, and only 1 of the 65 was identical
to Alcanivorax borkumensis. Members close to the Chromatia-
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TABLE 2. Summary of the most interesting 16S rRNA sequences detected from DGGE bands, clones, and degrading strains from OR and
Closest classified organism from
GenBank database (accession no.)
in phylogenetic treec
Alcanivoraceae (?) Alcanivorax spp. (AM286690, DQ347532,
3/65 3/72 3/29°Hx 30, 70
Chromatiales (?) Uncultured gammaproteobacterium
Uncultured soil bacterium clone M54-
R15?S11 4/65 1/7227
S8 1/65 4/72
Pseudomonadaceae (?) Pseudomonas spp. (AY691188, AJ312176)2/11*6/9*Phe 18, 45, etc.
Salinisphaeraceae (?) Salinisphaera sp. strain ARD M17
Xanthomonadaceae (?) Dokdonella koreensis DS-140 (AY987369)
Pseudoxanthomonas spadix IMMIBAFH-5
Stenotrophomonas sp. strain KL1A1
Hx (?)13, 72
SH8 Hx (?) 31, 71
Erythrobacteraceae (?)Erythrobacter sp. strain JL893 (DQ985055)R16?SH7 1/65 Hx (?)27, 40, 44, 50
Phyllobacteriaceae (?) Parvibaculum lavamentivorans (AY387398)R6/SH15/651/72 52, 53
Rhodobacteraceae (?) Uncultured Rhodobacteraceae bacterium
Citreicella sp. strain 2-2A (AB266065)
R9/R10/R111/65 Phe (?)K. Watanabe et al.,
Rhodospirillaceae (?) Thalassospira sp. strain DBT-2 (DQ659435) RH3 1/72B. Wang et al.,
Tistrella mobilis (AB071665)
Lutibacterium anuloederans (AY026916)
Novosphingobium spp. (AY690709,
Sphingomonas spp. (AY646154, AJ717392,
AY690679, AB099636, AF282616)
Sphingopyxis sp. strain FR1093 (DQ781321)
Sphingomonadaceae (?) 6/65
4/65 1/72 9/11* 3/9*Phe Y. Ahn, unp,; 22,
Flavobacteriaceae (B)Yeosuana aromativorans (AY682382) S131/72 34
Dietziaceae (A)Dietzia spp. (AB159036, X79290) 3/65 3/26°Hx11, 49, 74
Gordoniaceae (A) Gordonia polyisoprenivorans (DQ154925) 3/29°Hx 11, 39
Microbacteriaceae (A) Microbacterium spp. VKM Ac-2048
2/65 24, 51
Mycobacteriaceae (A)Mycobacterium spp. (AY255478, AJ276274,
5/728, 25, 32, 66
Nocardiaceae (A) Rhodococcus sp. strain 5/1 (AF181689)
Rhodococcus sp. strain MBIC01430
Rhodococcus opacus ML0004 (DQ474758)
9, 26, 46, 55
26, 46, 55
1/29° Hx 26, 46, 55
Williamsiaceae (A)Williamsia sp. strain MT8 (AY894336)R42/72
aDesignations and accession numbers for sequences and levels of similarity to related organisms are shown. Information from this study comes from isolated strains
that gave positive results in alkane or aromatic (Hx and Phe, respectively) degradation tests. Although it is not totally appropriate to draw conclusions about
physiological features from molecular data, references to other studies reporting any relationship of the sequence detected to biodegradation (degrading capacity,
detection in hydrocarbon-polluted samples. . .) are given.
b?, ?, and ? represent gamma-, alpha-, and deltaproteobacteria, respectively. B, Bacteroidetes; A, Actinobacteria; F, Firmicutes; P, Planctomycetes; C, Chloroflexi.
cSequences were matched with the closest relative from the GenBank database after a BLAST search and phylogenetic analysis.
dProportion of each degrading isolated species relative to the total number of isolates able to degrade the same fraction in each polluted matrix. Symbols: ?, identical
sequence; /, similar sequence; °, alkane-degrading isolates; *, aromatic-degrading isolates.
eCompilation of the available data from this and other studies about the degrading ability related to each 16S rRNA gene sequence. ?, related to alkane or aromatic
degradation but needs further study of its specific role; unp., unpublished data. Normally refers to the authors of the closest sequence in GenBank (accession number).
3412ALONSO-GUTIE´RREZ ET AL.APPL. ENVIRON. MICROBIOL.
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les group, the genera Parvibaculum and Lutibacterium, de-
tected in the DGGE profiles (see Tables S2 and S3 in the
supplemental material) were also found in the clone libraries
(7 to 12% of the clones; Table 2; see Table S4 in the supple-
mental material). The genus Salinisphaera (97 to 98% similar-
ity), although not detected by DGGE, constituted a high per-
centage of the library (9%). The Bacteroidetes group (6%) was
also found and was represented by four different genera (see
Table S4 in the supplemental material).
Oiled sand (OS) sample total community. A higher species
richness was found in OS than in OR samples (see Table S4 in
the supplemental material). The main bacterial groups were
the class Alphaproteobacteria (38% of the clones, including 20
different genera), the class Actinobacteria (30% of the clones,
including the genera Rhodococcus [8%] and Mycobacterium
[7%]), and the class Gammaproteobacteria (19% of the clones).
In contrast to OR samples, OS samples contained other
minor representatives such as members of the Deltapro-
teobacteria, Planctomycetes, and Chloroflexi groups. The Bac-
teroidetes group also presented a notable number of species
(see Table S4 in the supplemental material). Lutibacterium
anuloederans (96 to 99% similarity) clones, similar to bands
S10 and S6 (Fig. 4A), accounted for 6% of the clones. Most of
the clones related to Rhodococcus were again identical to
DGGE band S1 (99% similarity to R. fascians DSM20669).
Most of the members of the class Gammaproteobacteria were
close to sequences of uncultured Chromatiales and identical to
bands S8 (6%) and S9 (3%), while Alcanivorax was detected
again in low abundance (4%) and only 1 clone out of 72 was
identical to A. borkumensis.
Bacterial isolates. Alkane-degrading strains isolated from
the OR sample belonged exclusively to the genera Rhodococ-
cus and Dietzia of the class Actinobacteria (see Table S5 in the
supplemental material). In fact, 26 out of the 32 positive al-
kane-degrading strains matched exactly either the R1 ? S1 ?
RH1 or the R2 ? S2 ? RH2 band sequences from DGGE
belonging to the genus Rhodococcus (Fig. 6A), whereas 3 out
of 32 were related to the genus Dietzia. Rhodococcus type 1 and
2 strains were, respectively, identical to Rhodococcus sp. strain
5/1 (accession no. AF181689) and 99% similar to Rhodococcus
sp. strain MBIC01430 (accession no. AB088667) (Table 2).
Dietzia-related strains (e.g., PDR4 or PDR22), close to D.
maris (99 to 100% similarity) and D. psychralkaliphila (99 to
100% similarity), migrated close to Rhodococcus bands (Fig.
6A). Both belong to the class Actinobacteria, which is charac-
terized by a high G?C content and thus stability in its 16S
rRNA gene sequences, which migrated longer in the DGGE
gel. The different strains of Rhodococcus seemed to grow very
close in hexadecane culture, being indistinguishable and diffi-
cult to isolate. DGGE helped to detect those nonpure cultures
such as PDR23 which were a mixture of the two Rhodococcus
strains, types 1 and 2 (Fig. 6A). Strains were separated after-
ward with marine agar, where the different strains developed
different colors and morphologies. Although isolates mainly
from the enrichment cultures in hexadecane (PDA) also con-
firmed the dominance of Rhodococcus (16 out of 26; 80%
similarity), a higher number of additional species related to
alkane degradation (e.g., Gordonia, Erythrobacter, Stenotro-
phomonas, and Alcanivorax spp.; Fig. 6B; see Table S6 in the
supplemental material) could be isolated than from OR (Fig.
6A; see Table S5 in the supplemental material). Even though
isolates of both Erythrobacter (99% similar to bands SH7 and
R16 from the OS-Hx and OR DGGE profiles, respectively)
and Stenotrophomonas (identical to band SH8) were detected
in the population related to alkane degradation, no degrading
ability could be confirmed, in contrast to Dietzia, Rhodococcus,
Gordonia, and Alcanivorax isolates, which were able to grow on
hexadecane as the only source of C and energy (Table 2).
All isolates related to PAH degradation were obtained from
phenanthrene enrichments (PhR and PhS strains), like Sphin-
gomonas, Pseudomonas stutzeri, and Tistrella mobilis (Fig. 6C
and Table 2; see Table S1 in the supplemental material). One
exception occurred with Citreicella strain RP3, which could
only be isolated with one-fifth-strength marine agar directly
from fuel oil attached to rocks (OR). Aromatic-degrading abil-
ity could be confirmed only in species of two genera, Sphin-
gomonas and Pseudomonas. PAH-degrading strains of Sphin-
gomonas were close to DGGE bands of OS-PAHs (SP5-SP8),
FIG. 5. Cluster analysis from a similarity matrix generated from
DGGE profiles (Fig. 4) according to the Pearson product moment and
the unweighted-pair group method using average linkages. The DGGE
profile of OS samples was close to that of hydrocarbon degraders,
whereas the OR DGGE profile was more similar to that of alkane
degraders than to that of PAH degraders. NOS, nonoiled sand; OS
plus a number, oiled sand replicates; OR plus a number, oiled rock
replicates. OR-Hx, OR-PAH, OS-Hx, and OS-PAH, total DNA from
bacteria growing at the highest dilutions in hexadecane (Hx) and
aromatic (PAH) MPN analyses, respectively.
TABLE 3. Shannon-Weaver diversity indexes calculated for DGGE
profiles and numbers of DGGE bands detecteda
No. of DGGE
aFrom Fig. 4 and 6. NOS, nonoiled sand; OS plus a number, oiled sand
replicate; OR2, oiled rock replicate; OR-Hx, OR-PAH, OS-Hx, and OS-PAH,
total DNA from bacteria growing at the highest dilutions in hexadecane (Hx) and
aromatic (PAH) MPN analyses, respectively.
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and one of them was identical to bands RPb2 and SP7 (Table
2). Tistrella isolates had a sequence identical to DGGE band
RPb1 (OR-PAHs) and therefore are related to the degrada-
tion of aromatics, although no ability could be confirmed.
Something similar occurred with Citreicella strain RP3, which
had a 16S rRNA gene sequence close to DGGE bands R9 and
R10 and identical to band R11 and clone Rc10 (accession no.
EU375056) from OR samples (Table 2). The strain was close
(99 to 100% similarity) to Citreicella sp. strain 2-2A (accession
no. AB266065), although no degrading activity could be ob-
served in our strain with the methodology used.
Impact of fuel on microbial populations. The Prestige oil
spill did not affect bacterial abundances in the areas studied
but induced deep changes in the trophic structure of bacterial
communities. A similar situation was described after the Nak-
hodka oil spill, with a composition similar to that of the Prestige
fuel, in the marine communities of the Japan Sea (31, 43).
Although the communities were qualitatively different from
those in NOS, those affected by the oil spill still conserved high
species richness and diversity. These results agree with previ-
ous observations that community diversity was dramatically
reduced just after the pollution event and progressively recov-
ered to preoiling levels but with a different structure domi-
nated by hydrocarbonoclastic bacteria (24, 50). In the present
study, we observed that although rocks and sand are quite
different substrates, community compositions were quite sim-
ilar, suggesting that fuel oil drives the structure of the commu-
nities affected. However, the higher species richness and diver-
sity of OS communities detected by culture-dependent
and -independent methods suggests that the environmental
conditions on the OR surface, subject to daily contrasting
temperatures and dryness, may require a more specialized
microbial population to survive under such restricting condi-
tions compared to those that exist in sand, where a higher
number of different bacteria can grow.
Predominance of taxonomic groups and microbial diversity.
Most previous studies have focused on the short-term effects of
crude oil or its components on marine bacterial communities,
which usually became dominated by Gammaproteobacteria (1,
24, 50) just after an oil spill. In artificially oiled environments
amended with nutrients, biodegradation rates were promoted
and the first fast petroleum degradation processes were carried
out by communities dominated by Gammaproteobacteria (e.g.,
Alcanivorax, Cycloclasticus, Thalassolituus…), which were rap-
idly replaced by Alphaproteobacteria in less than a month (50).
In the present work, as previously done after the Nakhodka oil
spill in the Japan Sea (31), we focused on the analysis of
communities affected for a long time by heavy fuel oil. In the
affected coasts of the Japan Sea, where natural attenuation
proceeded slowly, probably due to the small amount of nutri-
ents present (total N, ?0.1 mg liter?1), bacterial communities
from oil paste were still dominated by Gamma- and Alphapro-
teobacteria (gram-negative) more than 12 months after the oil
spill (31), indicating that oil from the Nakhodka was still rich in
those more biodegradable fractions due to slow degradation
processes. However, Alphaproteobacteria and gram-positive
Actinobacteria dominated our oiled samples after the same
time. Gram-positive bacteria do not respond to high hydrocar-
bon inputs (42) and are never dominant just after an oil spill,
being detected in nonpolluted areas (33, 40) or in long-weath-
ered oil-polluted environments (48). The differences observed
FIG. 6. DGGE screening of isolated alkane-degrading strains from
rocks (A) and sand (B) and isolates related to PAH degradation from
both matrices (C). DGGE patterns of total communities (OR and OS)
and their respective hydrocarbon (Hx and PAH)-degrading popula-
tions were used as references to detect those strains playing roles in the
biodegradation of the Prestige crude oil in the environment (e.g., hexa-
decane-degrading strain PDR24, identical to bands R1 and RH1, in-
dicates the presence of Rhodococcus and its role in the in situ degra-
dation of the alkane fraction of the Prestige fuel). The OR, OS, OR-Hx,
OS-Hx, OR-PAH, and OS-PAH profiles, marked with arrows and bold
letters, were identical to those described in Fig. 4. Some of the se-
quenced bands are referenced again in the present figure to assist in
the detection of strains. The term “mix” indicate those isolates which
were mixtures of more than one strain (e.g., PDR23 was composed of
two strains of Rhodococcus, where the upper two bands were a het-
eroduplex of the two Rhodococcus 16S rRNA gene sequences).
3414ALONSO-GUTIE´RREZ ET AL.APPL. ENVIRON. MICROBIOL.
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between the molecular marker ratios of the original fuel oil
and those of the oiled samples (see Fig. S2 in the supplemental
material) are also consistent with the trends that follow bio-
degradation (28). The aromatic fraction exhibited a predomi-
nance of alkylnaphthalenes in the original oil that were almost
lost in the collected samples mainly by water washing and
evaporation (see Fig. S3B in the supplemental material). How-
ever, microbial degradation was observed as a severe depletion
of the n-alkane fraction, even the higher fraction (see Fig. S3A
in the supplemental material), and the relative reduction of
isomers with ? substituents such as the 2- and 3-methylphenan-
threnes and dibenzothiophenes within their respective series
(28). This enrichment in more recalcitrant fractions of the fuel
might explain the dominance of gram-positive bacteria previ-
ously hypothesized to have roles in the degradation of such less
biodegradable hydrocarbon classes (48).
Since the oil from the Nakhodka had a composition similar
to that from the Prestige (heavy fuel) and the time of sampling
was the same, it seems likely that the higher level of nutrients
(0.20 to 0.25 mg liter?1total N, of which 0.15 mg ml?1was
nitrate ) supplied to the littoral of the Costa da Morte
(northwestern Spain) by the northwestern Africa upwelling
system (20) is responsible for the observed differences in the
biodegradation rate (68) and community composition.
Alcanivorax (70) dominates oil-degrading communities when
nutrients are supplied, but with normal levels of nutrients more
diverse communities can exist (30, 50). In a recent study, we
reported the presence of A. borkumensis in high numbers just
after the Prestige oil spill in sediments of the Ría de Vigo (3),
where the alkane fraction was still abundant and higher nutri-
ent levels (0.6 mg liter?1total N) than at the Costa da Morte
(0.20 to 0.25 mg liter?1total N) existed (5). In this sense,
members of the well-described hydrocarbonoclastic genus Al-
canivorax were still present in OR and OS but in very low
numbers, as occurred in marine environments affected for long
times by heavy fuels (31, 48).
DGGE profile differences among the different trophic pop-
ulations detected and the reduction in the number of bands
with respect to the total profiles (Table 3) suggested an im-
portant specialization of species roles in the process of fuel
biodegradation in both matrices.
Population related to alkane degradation. Culture-indepen-
dent and -dependent analyses showed that Actinobacteria, mainly
Rhodococcus species, was the key alkane-degrading group of
bacteria. Rhodococcus has been associated with the degrada-
tion of n-alkanes up to C36(65) and branched alkanes (64),
which are particularly abundant in the Prestige fuel (16). It is
well known that Rhodococcus is a genus with remarkable met-
abolic diversity (36) and is able to produce biosurfactants
which can enhance not only the bioavailability of fuel compo-
nents but also the growth of other degrading bacteria (26, 47).
Dietzia and Microbacterium species, detected exclusively in the
OR clone library, have been, respectively, described as degrad-
ers of alkanes, including branched alkanes (49, 74), or related
to oil degradation in hydrocarbon-polluted sites (24, 51). Since
Dietzia, Microbacterium, and Rhodococcus belong to the class
Actinobacteria, some common characteristic might explain the
dominance of this group on OR. In this sense, an interesting
study which compared the different uptakes of hydrocarbons
by two Pseudomonas and Rhodococcus strains (59) clearly
showed how the hydrophobic surface developed by the latter
allowed the growth of Rhodococcus attached to the oil surface
increasing its degrading capacity. This capacity might explain
the relative major presence of Rhodococcus on OR compared
to OS since this ability could represent an important advantage
for survival in such a harsh environment.
Several members of the family Xanthomonadaceae that were
detected in this study were associated with the degradation of
alkanes since they were detected in Hx and PAH DGGE pro-
files (Fig. 4B and C and Table 2). In fact, with the exception of
D. koreensis, which was not previously related to either alkane
degradation or oil-polluted sites, the other species (P. spadix
and S. maltophilia) were previously associated with oil degra-
dation and surfactant production (13, 72). The alphapro-
teobacterium genus Erythrobacter, commonly encountered af-
ter first fast degradation processes (40, 50), was detected as
well as part of the population related to alkane degradation
(OS-Hx). Since no ability to degrade hydrocarbons could be
confirmed for any of these strains, they might play secondary
roles in the degradation of this fraction in collaboration with
Actinobacteria, mainly in OS, where a higher diversity existed
Populations related to aromatic degradation. The metabo-
lism of PAHs is a more complex process than the metabolism
of the aliphatic fraction, where the initial bacterial dioxygen-
ases from PAH metabolism exhibited a lower substrate speci-
ficity. Frequently, the resulting oxidized PAHs require the in-
tervention of another bacterial strain, which plays an important
role in degradation but cannot be detected as an aromatic
hydrocarbon degrader. In this sense, we described in a previous
work how the bacterial metabolism of fluorene needed the
coculture of two strains, of which only one was able to degrade
the aromatic while the other eliminated secondary metabolites
produced by the former (12). Probably this laboratory model
reproduces a very frequent metabolic cooperation among dif-
ferent strains in the bacterial metabolism of PAHs in situ.
Strains related to Sphingomonas were isolated as phenan-
threne-degrading strains in both matrices (Table 2). The aro-
matic-degrading Sphingomonas isolates in this study were quite
different from the single clone (Sc29) detected, and thus no
significance in the in situ degradation of the fuel mixture can
be attributed to these strains. However, two DGGE bands
(R12 and R17) and 4.5% of the OR library were related to
Sphingopyxis and Novosphingobium, respectively (Table 2), two
genera formerly considered to be Sphingomonas (57). More-
over, oil paste from a beach affected by the Nakhodka oil
spill presented sequences related to Sphingomonas subarc-
tica (100% similarity) which were proposed to play roles in
PAH degradation (31). Interestingly, other members of this
family could play a central role in the degradation of the
aromatic fraction of the Prestige fuel in both matrices, such as
L. anuloederans (95 to 100% similarity; two DGGE bands and
around 7% of the clones of each sample; Table 2). Although
we could not isolate any strain of this species, L. anuloederans
was described as a two- and three-ring PAH-degrading bacte-
rium which had a higher efficiency in the uptake of aromatics
than Cycloclasticus species do (14). Similar results have been
recently observed in a complete study performed in the
Thames estuary (United Kingdom), where Cycloclasticus
seemed to dominate seawater microcosms spiked with single
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PAHs except those containing fluorene, where a sequence
close to L. anuloederans was found (44).
Several clones close to different species of Mycobacterium
spp. were detected in OS. All of the clones were different from
each other, but some were close to the species Mycobacterium
frederiksbergense (98 to 100% similarity), which has previously
been reported to mineralize the PAHs phenanthrene, fluoran-
thene, and pyrene (66). Mycobacterium species are specialized
in the degradation of adsorbed PAHs in soils (8). However, the
most frequently used PAH-degrading bacterial isolation meth-
odology, including the one we used, is conducted with liquid
medium with agitation (7), so those strongly adhering bacteria
may tend to escape from conventional isolation techniques
(8, 60), as occurred in the present study. Fortunately, other
research groups could obtain aromatic-degrading isolates of
Mycobacterium spp. from pyrene enrichments of Prestige oil-
polluted samples which were really close to OS (M. Grifoll,
personal communication; http://otvm.uvigo.es/vertimar2007
Cycloclasticus has been proposed as the main PAH degrader
in many previous studies, including some done after the Nak-
hodka oil spill (43, 44). However, those studies analyzed com-
munities from seawater samples just after the oil spill at first
fast degradation processes when this and other Gammapro-
teobacteria, like Alcanivorax, dominated the community, while
the oiled matrices under study had already suffered from
weathering and biodegradation processes at the time of sam-
pling. The ability of L. anuloederans and Mycobacterium spp. to
degrade fluorene and pyrene, which are considered especially
recalcitrant fuel components (63), might explain the high
abundance of this bacteria in heavy fuel devoid of the most
easily biodegradable fractions.
T. mobilis was previously detected in seawater microcosms
spiked with PAH mixtures. In that case, it was hypothesized
that this species could have a secondary role in the degradation
of catabolic intermediates of aromatic compounds, owing to
their appearance only after 6 or 9 weeks of incubation (44). In
the present work, isolates of this strain could not grow as pure
isolates on PAHs with the methodology used. However, in
combination with the degrading Sphingomonas strain isolated,
a big growth of Tistrella could be observed. Although further
studies of the specific implication of T. mobilis in the aromatic
degradation process are needed, our observations suggest the
existence of a metabolic collaboration between them, where T.
mobilis probably grows on second metabolites derived from the
phenanthrene degradation carried out by Sphingomonas.
Strain RP3, detected by culture-dependent and -indepen-
dent methods on OR, was close (99 to 100% similarity) to
Citreicella sp. strain 2-2A (accession no. AB266065). This
strain, first isolated from seawater as a PAH-degrading bacte-
rium by Y. Kodama and K. Watanabe in 2006 (unpublished
results), was detected with the same sequence as an “uncul-
tured Roseobacter sp. (DQ870519)” in supralittoral rocks af-
fected by the Prestige oil spill more than 400 km from our
sampling site (27). This bacterium might need a cofactor avail-
able in the bacterial community of the oiled cobblestones to
develop its degrading capacity since no growth was observed in
the aromatic degradation test of RP3 in mineral medium with
Ubiquity of bacterial species. Sequences close to Rhodococ-
cus, Chromatiales, Rhodobacteraceae, Roseobacter (Citreicella),
and Erythrobacter detected in both of the samples under study
(OR and OS) were, respectively, identical to the sequences
with accession numbers DQ870544, DQ870518, DQ870525,
DQ870519, and DQ870538 retrieved from another cobble-
stone beach affected by the Prestige spill (27). In addition,
several sequences found in our clone libraries showed at least
99% similarity to other DGGE bands detected in that study.
What is more interesting is that DGGE profiles from that study
became more similar to those of our OR and OS samples at
advanced stages of the degradation process (27), which agrees
with our hypothesis. Although samples were taken from rock
surfaces similar to our OR, the beach was more than 400 km
from our sampling point. Therefore, it seems that conclusions
derived from the present work can be applied to other parts of
the Spanish coast affected by the Prestige oil spill.
Bioremediation amendments. Mycolic acids, very-long-chain
(C30to C90) ?-alkyl, ?-hydroxy fatty acids, are major and spe-
cific constituents of a distinct group of gram-positive bacteria,
classified in the suborder Corynebacterineae, which includes
genera detected in the present study such as Mycobacterium,
Williamsia, Gordonia, Dietzia, and Rhodococcus. As opposed to
gram-negative bacteria, such Pseudomonas or Alcanivorax, that
dominate fast petroleum degradation processes at first (30),
members of this group are never dominant at such stages (42,
48), being detected with higher frequency in resource-limited
environments, where they could play a key role in the in situ
degradation of more recalcitrant components a long time after
an oil spill (48). Unusually, these gram-positive bacteria con-
tain an outer permeability barrier that may explain both the
limited permeability of their cell walls and their general non-
susceptibility to toxic agents (21), which has been related to an
enhanced biodegradation capacity (37, 38). Therefore, the ad-
dition of mycolic acids to bioremediation amendments applied
to coasts with ecological features close to those of the affected
Spanish areas and affected for a long time by a contaminant
similar to the Prestige fuel, might be a good strategy to enhance
in situ degradation.
Conclusions. This study shows that supralittoral areas with
favorable environmental conditions and polluted with heavy
fuels are likely to be dominated, after some months of weath-
ering and biodegradation processes, by Actinobacteria (mainly
of the suborder Corynebacterineae) since this group, character-
ized by long-term survival in the environment even under dry,
resource-limited conditions, might degrade the more recalci-
trant fractions of the remaining fuel. Since the use of already
acclimated indigenous microorganisms is always preferable to
the use of externally inoculated degraders, the addition of
mycolic acids to favor the activity of autochthonous Coryne-
bacterineae is proposed for bioremediation amendments ap-
plied at later stages of bioremediation.
This research was supported by project VEM 2003-20068-C05-01 of
the Spanish Ministerio de Educacio ´n y Ciencia. J.A. and N.J. thank the
Ministerio de Educacio ´n y Ciencia for their predoctoral fellowships.
We also thank M. A. Murado, J. Miro ´n, and F. J. Fraguas from the
Departamento de Reciclado y Valoracio ´n de Residuos of IIM (CSIC-
Vigo) for their support in sampling site localization and X. A. A ´lvarez-
3416 ALONSO-GUTIE´RREZ ET AL.APPL. ENVIRON. MICROBIOL.
at CSIC-PUBLICACIONES ELECTRONICAS on June 11, 2010
Salgado for the interesting discussion of nutrient levels at the Costa da
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