Blackwell Science, LtdOxford, UKEMIEnvironmental Microbiology 1462-2920Blackwell Publishing Ltd, 200359746753Original ArticleAlcanivorax – substrate specificity for alkane degradationA.

Hara, K. Syutsubo and S. Harayama

Environmental Microbiology (2003) 5(9), 746–753 doi:10.1046/j.1462-2920.2003.00468.x

Alcanivorax which prevails in oil-contaminated

seawater exhibits broad substrate specificity for
alkane degradation

Akihiro Hara,*† Kazuaki Syutsubo and to coastal fauna and flora. Upon the release of oil in the
Shigeaki Harayama natural environment, the transformation of its constituents
Marine Biotechnology Institute, Kamaishi Laboratories, starts immediately, and microbial degradation is consid-
3-75-1 Heita, Kamaishi, Iwate 026–0001, Japan. ered to be a major route for the breakdown of hydrocarbon
components. Indeed, many bacteria that are capable of
degrading petroleum hydrocarbons have been isolated
Summary
from seawater. However, their roles in the biodegradation
Alcanivorax is an alkane-degrading marine bacterium of spilled oil in the natural marine environment have
which propagates and becomes predominant in remained unknown.
crude-oil-containing seawater when nitrogen and Alcanivorax strains, which are members of the g-
phosphorus nutrients are supplemented. In order to subclass of the Proteobacteria that can utilize n-alkanes
understand why Alcanivorax overcomes other bacte- as the sole sources of carbon and energy, have recently
ria under such cultural conditions, competition exper- been isolated from various seawater samples (Abraham
iments between Alcanivorax indigenous to seawater et al., 1998; Yakimov et al., 1998; Harayama et al., 1999).
and the exogenous alkane-degrading marine bacte- Although these Alcanivorax strains can utilize various ali-
rium, Acinetobacter venetianus strain T4, were con- phatic hydrocarbons as the sole sources of carbon and
ducted. When oil-containing seawater supplemented energy, they cannot use amino acids and carbohydrates
with nitrogen and phosphorus nutrients was inocu- (Yakimov et al., 1998; Dutta and Harayama, 2001). More
lated with A. venetianus strain T4, this bacterium was interestingly, they become the predominant microbial
the dominant population at the early stage of culture. community established in oil-contaminated seawater
However, its density began to decrease after day 6, when nitrogen and phosphorus nutrients are supple-
and Alcanivorax predominated in the culture after day mented (Harayama et al., 1999; Kasai et al., 2001; 2002;
20. The crude-oil-degrading profiles of both bacteria Syutsubo et al., 2001). Thus, Alcanivorax strains seem to
were therefore investigated. Alcanivorax borkumen- play an important role in the biodegradation of petroleum
sis strain ST-T1 isolated from the Sea of Japan exhib- hydrocarbons in an oil-polluted marine environment. How-
ited higher ability to degrade branched alkanes ever, the reason why bacteria belonging to Alcanivorax
(pristane and phytane) than A. venetianus strain T4. It become predominant in oil-contaminated seawater has
seems that this higher ability of Alcanivorax to not yet been elucidated.
degrade branched alkanes allowed this bacterium to We have isolated in a previous study several oil-degrad-
predominate in oil-containing seawater. It is known ing bacteria from various marine environments including
that some marine zooplanktons produce pristane and Acinetobacter venetianus strain T4 (MBIC 01332), Rhodo-
Alcanivorax may play a major role in the biodegrada- coccus erythropolis strain PR4 (MBIC 01337), Pseudomo-
tion of pristane in seawater. nas putida strain PB4 (MBIC 01340) and Sphingomonas
xenophaga strain AJ1 (MBIC 01552). Acinetobacter vene-
tianus strain T4 was capable of utilizing various n-alkanes
Introduction
as growth substrates, and grew to the highest density in
Petroleum hydrocarbons are an important energy an artificial consortium composed of these four strains
resource used by industry and in our daily life. At the same (Komukai-Nakamura et al., 1996).
time, petroleum is a major pollutant of the marine environ- We studied in this present work the growth in seawater
ment: tanker accidents cause serious ecological damage of indigenous Alcanivorax strains in competition with
exogenous A. venetianus strain T4, as this would reveal
Received 29 January, 2003; accepted 31 March, 2003. *For corre- the mechanism by which Alcanivorax predominates in oil-
spondence. E-mail akhara@sumitomopharm.co.jp; Tel. (+81) 66466 contaminated seawater. We then applied two molecular
5284; Fax (+81) 66466 5491. †Present address (from October 2002):
Sumitomo Pharmaceuticals Co. Ltd, Genomic Science Laboratories, methods, fluorescence in situ hybridization (FISH) and
3-1-98 Kasugade Naka, Konohana-ku, Osaka 554–0022, Japan. denaturing gradient gel electrophoresis of PCR-amplified
© 2003 Society for Applied Microbiology and Blackwell Publishing Ltd
 Alcanivorax – substrate specificity for alkane degradation 747
16S rDNA fragments (PCR/DGGE), to identify and quan-
tify these hydrocarbon-degrading bacteria. Based on the
results obtained, we concluded that the broad specificity
of petroleum hydrocarbon biodegradation in Alcanivorax
was one of factors which caused its dominant growth in
oil-contaminated seawater.
Results
Growth competition between Alcanivorax and
A. venetianus strain T4 in oil-contaminated seawater
In order to elucidate the mechanism underlying the pref-
erential growth of Alcanivorax, the growth competition
between indigenous Alcanivorax in seawater and exo-
genous A. venetianus strain T4 was examined. Fifty mil-
lilitres of non-sterilized seawater in a 500 ml Erlenmeyer
flask were supplemented with nitrogen, phosphorus
nutrients and crude oil and A. venetianus strain T4 was
used to inoculate the seawater at a density of 5 ¥ 103
cells ml-1. The cell density of indigenous Alcanivorax in
this seawater sample was estimated to be about 10
cells ml-1 by the MPN method (data not shown). This
number is in agreement with the density of indigenous
Alcanivorax in other fresh seawater samples determined
by the competitive PCR method (H. Kishira and S.
Harayama, manuscript in preparation). It was therefore
calculated that A. venetianus strain T4 was added to the
culture to a density approximately 500-fold higher than
that of indigenous Alcanivorax. The culture was incubated
at 20∞C with rotary shaking operated at 100 r.p.m. One
week after starting the cultivation, the total direct count
reached 2 ¥ 108 cells ml-1 (Fig. 1). The cell numbers of
Alcanivorax and A. venetianus strain T4 were, respectively,
determined by FISH. At an early stage of the culture, A.
venetianus strain T4 grew rapidly and predominated in the
culture (Figs 1 and 2). However, the cell number of A.
venetianus strain T4 began to decrease by day 6. In con-
trast, the cell number of Alcanivorax increased with
increasing incubation time. Alcanivorax finally predomi-
nated in the culture, its cell number reaching to 6 ¥ 107
cells ml-1 on day 21 (Figs 1 and 2) and accounting for 60%
of the total bacteria. Similar experiments were carried out
three times, and Alcanivorax always dominated the micro-
bial communities after prolonged incubation for three
weeks or more even when A. venetianus strain T4 was
added to a density approximately 500-fold higher than that
of indigenous Alcanivorax (data not shown).
Growth of A. venetianus strain T4 in spent medium from
a culture of A. borkumensis strain ST-T1
It was reported that Alcanivorax bacteria produced biosur-
factants during cultivation on n-alkane. (Yakimov et al.,
1998). Therefore, the possibility that substances produced
© 2003 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 5, 746–753
Fig. 1. Time-course characteristics of the growth of indigenous Alca-
nivorax bacteria (circles) and exogenous A. venetianus strain T4
(squares) on crude oil. Cells of A. venetianus strain T4 were use to
inoculate at a density of 5 ¥ 103 cells ml-1 in 50 ml of the NSW
medium containing crude oil (1 g l-1). Incubation was done at 20∞C
on a rotary shaker (100 r.p.m.). Total direct counts (triangles) were
determined by DAPI staining. Cell numbers of Alcanivorax bacteria
(circles) and A. venetianus strain T4 (squares) were, respectively,
determined by FISH with the ALV735 and ACA probes. Each data
point and error bar represents the mean and standard deviation,
respectively, of triplicate samples from a single experiment. Three
independent experiments were carried out, and the predominant
growth of Alcanivorax was always apparent.
by Alcanivorax inhibit the growth of A. venetianus strain
T4 was investigated. Strains closely related to the type
strain of the genus Alcanivorax, A. borkumensis strain
SK2T, have generally been isolated from cultures predom-
inated by Alcanivorax (H. Kishira and S. Harayama, manu-
script in preparation). Alcanivorax borkumensis strain
ST-T1 which is one of such strains was grown in the
liquid synthetic medium (BSM) supplemented with n-
hexadecane (1 g l-1) in a 500-ml Erlenmeyer flask. After
4 days, the medium from the saturated culture was steril-
ized by filtration, and the filtered medium was supple-
mented with n-hexadecane (1 g l-1) to prepare ‘spent
medium’. Acinetobacter venetianus strain T4 or Alcanivo-
rax borkumensis strain ST-T1 was used to inoculate the
spent media with at a density of 2 ¥ 107 cells ml-1. Two
days after starting the cultivation, the total direct count of
A. venetianus strain T4 reached 5.1 ¥ 108 cells ml-1. At
that time, the total direct count of A. borkumensis strain
ST-T1 reached 5.5 ¥ 108 cells ml-1. Acinetobacter vene-
tianus strain T4 grew rapidly as well as A. borkumensis
strain ST-T1, and the growth of A. venetianus strain T4
was not inhibited in such spent medium. Therefore, it
seemed that A. borkumensis strain ST-T1 did not produce
the toxic products.
Crude oil degradation profiles of A. borkumensis strain
ST-T1 and A. venetianus strain T4
Crude oil contains thousands of hydrocarbons and related
748 A. Hara, K. Syutsubo and S. Harayama
compounds (Rossini, 1960). Obtained results in the com-
petition experiment suggested the possibility that indige-
nous Alcanivorax could utilize a broader range of
hydrocarbon substances in crude oil than could A. vene-
tianus strain T4. The oil degradation profiles of A. borku-
mensis strain ST-T1 and A. venetianus strain T4 were
investigated by TLC-FID. Two weeks after starting the
cultivation, A. venetianus strain T4 had degraded about
44% of the saturated fraction of crude oil, whereas A.
borkumensis strain ST-T1 had degraded about 63% of the
© 2003 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 5, 746–753
Fig. 2. FISH with the ALV735 and ACA probes.
A. FISH with the ACA probe applied to a bac-
terial community established in the experiment
shown in Fig. 1 on day 4.
B. FISH with the ALV735 probe applied to the
day 4 bacterial community.
C. FISH with the ACA probe applied to the day
21 bacterial community.
D. FISH with the ALV735 probe applied to the
day 21 bacterial community. Hybridization was
performed as described in the Experimental
procedures section. DAPI-stained (left) and
probe-hybridized (right) photomicrographs of
identical fields are shown for each sample.
saturated fraction. It seemed that A. borkumensis strain
ST-T1 degraded the saturated fraction more efficiently
than did A. venetianus strain T4. The biodegradation pro-
files of n- and branched alkanes were also investigated
by GC/MS. Crude oil contains various branched alkanes,
pristane (2,6,10,14-tetramethylpentadecane) and phytane
(2,6,10,14-tetramethylhexadecane) being the most abun-
dant species. Although both bacteria degraded n-alkanes
almost completely (data not shown), the degradation abil-
ity for branched alkanes was very different between these
 Alcanivorax – substrate specificity for alkane degradation 749
two bacteria. Alcanivorax borkumensis strain ST-T1
degraded pristane and phytane almost completely,
whereas A. venetianus strain T4 degraded these com-
pounds at a level of less than 50%. It was considered from
these results that the ability to degrade branched alkanes
might be an important factor for growth on crude oil under
prolonged cultivation conditions.

Growth of A. borkumensis strain ST-T1 and A. venetianus

strain T4 on n-hexadecane and pristane

The growth of Alcanivorax borkumensis strain ST-T1 and

A. venetianus strain T4 on n-hexadecane and pristane in
BSM was examined by determining the total direct counts
of bacterial cells (Fig. 3). A time lag was observed in the
growth of A. borkumensis strain ST-T1 on n-hexadecane,
such that A. venetianus strain T4 grew faster than A.
borkumensis strain ST-T1 on this compound. In contrast,
A. borkumensis strain ST-T1 grew faster than A. vene-
tianus strain T4 on pristane. It seems that A. borkumensis
strain ST-T1 preferred pristane to n-hexadecane while it
was vice versa for A. venetianus strain T4.

Growth of Alcanivorax in seawater containing crude oil,

n-hexadecane or pristane

The growth of indigenous Alcanivorax in the NSW medium

containing crude oil, n-hexadecane, or pristane was
examined by determining the cell density of Alcanivorax
by FISH. The total direct count reached 108 cells ml-1 in
the medium containing crude oil or pristane (Fig. 4A),
while this value reached only about 2 ¥ 107 cells ml-1 in
the medium containing n-hexadecane. The degree of bac-
terial growth in the medium containing n-hexadecane was
lower than that in the medium containing crude oil or
pristane. On day 21, the size of the Alcanivorax population
determined by FISH was about 60% on the crude oil,
about 1% on n-hexadecane and about 80% on pristane. Fig. 3. Growth of A. borkumensis strain ST-T1 (circles) and A. vene-
The bacterial community structure in each of these cul- tianus T4 (squares) on n-hexadecane (A) and pristane (B). Ten mil-
tures was also examined. DNA was extracted from each lilitres of the BSM medium in a test tube containing n-hexadecane
(1 g l-1) or pristane (1 g l-1) were inoculated with 5 ¥ 105 cells/ml of A.
culture, the V3 region of small-subunit ribosomal RNA borkumensis strain ST-T1 or A. venetianus T4. The test tube was
genes was PCR-amplified from the DNA sample, the reciprocally shaken at 90 strokes per min at 20∞C. Total direct count
amplified products were analysed by DGGE, and some of was determined by DAPI staining. Data points and error bars
represent the mean and standard deviation of three independent
the major DGGE bands were excised and sequenced. The experiments.
sequences were then analysed for similarity to previously
obtained sequences in databases. Three independent
experiments were carried out, one of the results being omonas and Roseobacter were detected as the major
shown in Fig. 4B. The sequences in the databases exhib- bacteria in this culture with n-hexadecane.
iting the greatest similarity to the sequences of the DGGE
bands in Fig. 4B are shown in Table 1. In the presence of
PCR/DGGE analysis of the bacterial communities grown
crude oil and pristane, the most intensive band corre-
in seawater containing various alkanes
sponded to the Alcanivorax sequence. In contrast, the
band corresponding to the Alcanivorax sequence was not The bacterial communities established in cultures grown
detected in the culture with n-hexadecane. Instead, Hyph- on various alkanes were analysed by PCR-DGGE (Fig. 5),

© 2003 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 5, 746–753
750 A. Hara, K. Syutsubo and S. Harayama
other. Alcanivorax was only predominant in the cultures
grown on pristane and crude oil (Table 1). Squalane
(2,6,10,15,19,23-hexamethyltetracosane) is a branched
long-chain alkane (Berekaa and Steinbuchel, 2000), and
Alcanivorax was the major population in the NSW medium
containing 1 g l-1 of squalane (data not shown). This result

Table 1. 16S rDNA sequences most closely related to those of the

major bacterial populations detected by PCR/DGGE.

DGGE
band Closest GenBank relative Homology Accession No.

A1 Alcanivorax borkumensis 99% Y12579

A2 Hyphomonas species 99% M83812
A3 Uncultured bacterium 95% AF143825
A4 Roseobacter sp. KT0202a 97% AF305498
A5 Roseobacter sp. KT0202a 98% AF305498
A6 Uncultured bacterium 98% AF143825
B1 Cytophagales str. 96% AF025553
B2 Alcanivorax borkumensis 99% Y12579
B3 Unclassified Pseudomonas 96% AF102866
B4 Uncultured bacterium 98% AF128781
B5 Uncultured proteobacterium 96% AF191762

Fig. 4. Growth of bacteria indigenous to seawater on n-hexadecane,

pristane or crude oil (A), and 16S rDNA DGGE profiles of the grown
bacteria (B). The NSW medium (300 ml) supplemented with n-hexa-
decane (0.1 g l-1), pristane (0.1 g l-1) or crude oil (0.1 g l-1) was incu-
bated at 20∞C on a rotary shaker (100 r.p.m.). The growth of bacteria
on n-hexadecane (triangles), pristane (circles) or crude oil (squares)
was monitored by DAPI staining. Each data point and error bar
represents the mean and standard deviation, respectively, of three
independent experiments. An aliquot of each culture was sampled for
a PCR/DGGE analysis on day 14.
Fig. 5. 16S rDNA DGGE profiles of cultures grown on different
n-octane, n-dodecane, n-hexadecane, pristane and crude n-alkanes. The NSW medium (300 ml) containing n-octane,
oil being used as growth substrates. It is clear that the n-dodecane, n-hexadecane, pristane or crude oil at a concentration
of 1 g l-1 was incubated at 20∞C on a rotary shaker (100 r.p.m.). After
structures of the bacterial communities in the cultures with 3 weeks, an aliquot of each culture was sampled. PCR/DGGE was
different alkanes were completely different from each carried out as described in the Experimental procedures section.

© 2003 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 5, 746–753
 Alcanivorax – substrate specificity for alkane degradation 751
also shows that Alcanivorax has particular ability for
branched-alkane biodegradation in seawater.
Discussion
Crude oil contains various hydrocarbons and related com-
pounds, its major constituents being classified into the
saturated and aromatic fractions. The saturated fractions
contain n-, branched, and cyclo-alkanes. In general, bac-
teria preferentially use n-alkanes for growth. Although
Alcanivorax degraded n-alkanes, it was apparent that the
growth of Alcanivorax bacteria on n-alkanes was less
rapid than that of A. venetianus strain T4. The reason why
Alcanivorax became predominant in marine bacterial con-
sortia grown in crude-oil-contaminated seawater was
therefore not due to its ability to grow on n-alkanes. The
results obtained in this report indicate the possibility that
its excellent ability to degrade branched alkanes is one of
factors which allowed this strain to predominate in oil-
contaminated seawater.
As already described, n-alkanes are preferentially
degraded over branched alkanes by most alkane-degrad-
ing bacteria (Pirnik et al., 1974). It was demonstrated for
A. venetianus strain T4 that the speed of degradation of
branched alkanes was generally slower than that of n-
alkanes. Moreover, it has been reported that the degrada-
tion of branched alkanes was inhibited by n-alkanes with
the pristane-degrading bacterium, Brevibacterium eryth-
rogenes (Pirnik et al., 1974). Thus, branched alkanes are
recalcitrant to biodegradation, and phytane and pristane
have often been used as biomarkers which are relatively
unchanged petroleum components, even after extensive
biodegradation. We have demonstrated here that Alca-
nivorax grew on pristane much faster than A. venetianus
strain T4, and that pristane degradation was not inhibited
by n-alkanes with Alcanivorax (data not shown). These
properties of Alcanivorax allowed this bacterium to grow
continuously, even after the exhaustion of readily biode-
gradable n-alkane components.
Pristane is a ubiquitous substance in seawater, and
zooplanktons belonging to the genera Calanus and Neo-
calanus produce pristane (Avigan and Blumer, 1968)
which is transported from its sources into the lobster,
fish and whale. While some pristane-degrading bacteria
have been reported (McKenna and Kallio, 1971; Pirnik
et al., 1974), their role in the degradation of pristane in
a marine ecosystem remains unknown. We originally
found Alcanivorax to be an oil-degrading marine
bacterium exhibiting broad specificity to petroleum
constituents, including n- and branched alkanes, n-
alkylcycloalkanes and n-alkylbenzenes (Dutta and
Harayama, 2001). However, as pristane is ubiquitous in
seawater, the primary role of Alcanivorax in the marine
ecosystem may be to degrade pristane. This inference
© 2003 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 5, 746–753
could explain why Alcanivorax is widely distributed in the
marine environment.
Experimental procedures
Bacterial strains
Alcanivorax borkumensis strain ST-T1 (MBIC 04326) was
isolated from a seawater sample taken from the Sea of Japan
(Dutta and Harayama, 2001; Syutsubo et al., 2001). The n-
alkane-degrading bacterium, A. venetianus strain T4, has
been previously described (Komukai-Nakamura et al., 1996).
Details of these strains are available online from Marine
Biotechnology Institute Culture Collection (MBIC; http://
www.mbio.jp/mbic/).
Culture conditions
The bacteria were routinely maintained on one-fifth strength
Marine Broth 2216 (Difco) plates supplemented with 1.5%
(wt/vol) agar. Cultivation of the bacteria in synthetic liquid
media was carried out at 20∞C in a basal salt medium (BSM)
(Goto et al., 1994) supplemented with an appropriate hydro-
carbon substrate. Natural seawater was collected from Heita
bay (Kamaishi, Iwate, Japan) from a depth of 15 m. This
seawater was supplemented with NH4Cl and KH2PO4 to con-
centrations of 20 mg of N l-1 and 10 mg of P l-1, respectively,
to prepare the natural seawater (NSW) medium. Arabian light
crude oil was distilled at 230∞C to remove the volatile and
readily biodegradable components, and the unevaporated
part was used in this study (Dutta and Harayama, 2001). The
initial concentration of crude oil included in each medium was
1 g l-1. The bacterial growth in each culture was followed
by determining the total direct count using fluorescence-
microscopy, after staining with 4,6-diamidino-2-phenylindole
(DAPI), as described previously (Watanabe et al., 1998).
Most-probable-number (MPN) method for estimating the
density of Alcanivorax in the seawater samples
The cell density of Alcanivorax in a seawater sample was
estimated by the most-probable-number (MPN) method.
Serial 10-fold dilutions of a natural seawater sample in the
BSM medium were prepared, and one-millilitre aliquots of
each dilution were transferred to 10 replicate tubes each
containing 9 ml of the BSM medium supplemented with crude
oil (1 g l-1). The tubes were incubated at 20∞C for two weeks,
and aliquots of the culture were plated on one-fifth strength
Marine Broth 2216 (Difco) plates. The plates were subse-
quently incubated at 20∞C for one week, and the development
of Alcanivorax on the plates was examined. Colonies of Alca-
nivorax could be easily identified by their morphology as
Alcanivorax formed transparent colonies on these plates.
Most-probable-number values were calculated by using the
tables of Rowe et al. (1977).
Fish
The FISH method was performed as described previously
752 A. Hara, K. Syutsubo and S. Harayama
(Syutsubo et al., 2001). Cells of Alcanivorax were detected
with an ALV735 probe (5¢-CGTCAATGTCAGTCCAGGAGG-
3¢, E. coli 16S rDNA positions 735–750) that had been mono-
labelled with tetramethyl rhodamine isothiocyanate (TRITC)
at the 5¢ end, whereas those of A. venetianus strain T4 were
detected with a TRITC-labelled ACA probe (5¢-ATCCTCTC
CCATACTCTA-3¢, E. coli 16S rDNA positions 652–669)
(Wagner et al., 1994). These probes purified by high-
pressure liquid chromatography were purchased from Takara
Shuzo. Microscopic observations were carried out in at least
five fields for each well with a fluorescence microscope (BX-
60, Olympus) equipped with a cooled charge-coupled device
(CCD) camera system (PXL1400, Photometrics). Over 1000
DAPI-stained cells were counted to determine the ratio of the
probe-labelled cells to the total cells.
Assays for biodegradation of the hydrocarbons and
crude oil
Cells of A. borkumensis strain ST-T1 or A. venetianus strain
T4 were used to inoculate 10 ml of the BSM medium in a test
tube containing crude oil (1 g l-1) at a density of 5 ¥ 105
cells ml-1. The test tube was reciprocally shaken at 90 strokes
per min and at 20∞C for 14 days. The degradation rates for
pristane and phytane were determined by the GC/MS. After
the incubation, the crude oil remaining in each test tube was
extracted with dichloromethane and then evaporated at 50∞C.
Samples thus obtained were dissolved in a determined vol-
ume of chloroform, before being subjected to further analyses.
The degree of biodegradation of the saturated, aromatic, resin
and asphaltene fractions of the crude oil was determined by
the Iatroscan M-5 system (Iatron Laboratories, Tokyo) as
described previously (Goto et al., 1994); this system utilizes
thin-layer chromatography in combination with flame ioniza-
tion detection (TLC/FID). Analyses of the hydrocarbon
components in crude oil were also carried out by gas
chromatography/mass spectrometry (GC/MS) with a Shi-
madzu QP-5000 instrument as described previously (Wang
et al., 1994; Maki et al., 1999). All values obtained were nor-
malized to that of 17a(H), 21b(H)-hopane (Prince et al., 1994).
PCR/DGGE
Cells in the liquid cultures were collected by centrifugation
and suspended in a TE buffer, before DNA was extracted
from the cells according to the method of Marmur (Marmur,
1961). Polymerase chain reaction/DGGE was performed as
described previously (Watanabe et al., 1998). Polymerase
chain reaction primers P2 and P3 (Muyzer et al., 1993) were
used to amplify the variable V3 region of bacterial 16S rDNA
(corresponding to positions 341–534 in the Escherichia coli
rDNA sequence) connected to a GC clamp. DNA sequences
of the DGGE bands were determined and compared with the
sequences of 16S rDNA genes available from the GenBank
by performing a BLAST search (Altschul et al., 1990) through
the US National Institute of Health’s Internet site.
Acknowledgements
We thank Mr Hideo Kishira for valuable discussions, and we
© 2003 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 5, 746–753
are grateful to Ms. Satomi Takahashi for her technical assis-
tance. This work was supported by the New Energy and
Industrial Technology Development Organization (NEDO).
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