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Applied and Environmental Microbiology, December 1999, p. 5532-5540, Vol. 65, No. 12
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Initial Reactions in Anaerobic Alkane
Degradation by a Sulfate Reducer, Strain AK-01
Chi Ming
So
and
L. Y.
Young*
Biotechnology Center for Agriculture and the
Environment and Department of Environmental Sciences, Cook College,
Rutgers, The State University of New Jersey, New Brunswick, New Jersey
08901-8520
Received 28 June 1999/Accepted 28 September 1999
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ABSTRACT |
An alkane-degrading, sulfate-reducing bacterial strain, AK-01,
isolated from a petroleum-contaminated sediment was studied to
elucidate its mechanism of alkane metabolism. Total cellular fatty
acids of AK-01 were predominantly C even when it was grown on C-even
alkanes and were predominantly C odd when grown on C-odd alkanes,
suggesting that the bacterium anaerobically oxidizes alkanes to fatty
acids. Among these fatty acids, some 2-, 4-, and 6-methylated fatty
acids were specifically found only when AK-01 was grown on alkanes, and
their chain lengths always correlated with those of the alkanes. When
[1,2-13C2]hexadecane or perdeuterated
pentadecane was used as the growth substrate, 13C-labeled
2-Me-16:0, 4-Me-18:0, and 6-Me-20:0 fatty acids or deuterated 2-Me-15:0, 4-Me-17:0, and 6-Me-19:0 fatty acids were recovered, respectively, confirming that these monomethylated fatty acids were
alkane derived. Examination of the 13C-labeled 2-, 4-, and
6-methylated fatty acids by mass spectrometry showed that each of them
contained two 13C atoms, located at the methyl group and
the adjacent carbon, thus indicating that the methyl group was the
original terminal carbon of the
[1,2-13C2]hexadecane. For perdeuterated
pentadecane, the presence of three deuterium atoms, on the methyl group
and its adjacent carbon, in each of the deuterated 2-, 4-, and
6-methylated fatty acids further supported the hypothesis that the
methyl group was the terminal carbon of the alkane. Thus, exogenous
carbon appears to be initially added to an alkane subterminally at the
C-2 position such that the original terminal carbon of the alkane
becomes a methyl group on the subsequently formed fatty acid. The
carbon addition reaction, however, does not appear to be a direct
carboxylation of inorganic bicarbonate. A pathway for anaerobic
metabolism of alkanes by strain AK-01 is proposed.
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INTRODUCTION |
Alkanes are major components of
petroleum fuels commonly found in polluted environments, and numerous
studies of their biodegradability by microorganisms have been
conducted. Most of the earlier studies focused on processes that take
place under aerobic conditions (6), in which alkanes are
initially oxidized by oxygen to alcohols by monooxygenases, with
detailed accounts of the biochemistry and genetics (37, 38).
In contrast, while alkane degradation under anaerobic conditions has
been reported in early as well as recent literature (2, 7, 9, 27,
32), much less is understood about the mechanism(s) of
degradation in the absence of oxygen. Some reports indicated that
alkanes could be enzymatically dehydrogenated to alkenes (8,
19), and the reaction may have served as an initial reaction for
anaerobic alkane degradation (29). These observed
activities, however, could not be shown to support growth under
anaerobic conditions (17).
In more recent studies, Aeckersberg et al. isolated and characterized a
sulfate-reducing, alkane-degrading strain, Hxd3 (2). We have
also recently reported the isolation and characterization of an
alkane-degrading strain, AK-01, which is different from Hxd3
phylogenetically (35). Hxd3 appears to anaerobically oxidize alkanes to fatty acids, as evidenced by the influence of alkane carbon
chain lengths on its cellular fatty acid composition (3). Other observations also suggest that an odd-numbered carbon atom(s) may
be initially added to or removed from the alkane chain terminus before
the fatty acids are formed. On the other hand, evidence suggests that
desaturation of the alkane to the corresponding 1-alkene, a
hypothetical initial step previously proposed in the literature
(29), does not occur during anaerobic alkane oxidation by
Hxd3 (1, 3). Another alkane-degrading strain, Pnd3, reported by the same authors (3) also appears to anaerobically
oxidize alkanes to fatty acids, though seemingly through a different
initial reaction(s) (3, 14). These initial reactions are not
yet understood.
In this study, we investigated the initial reaction(s) of alkane
degradation by strain AK-01. This strain, which was isolated from a
petroleum-contaminated estuarine sediment, is able to oxidize alkanes
completely to carbon dioxide, with coupling to sulfate reduction
(35). Early evidence also suggests that it anaerobically oxidize alkanes to fatty acids. By using stable- isotope-labeled alkanes as growth substrates, we clearly documented that alkanes are
oxidized to fatty acids by this organism. Structural characterizations of the isotope-labeled fatty acids derived from the labeled alkanes demonstrated that exogenous carbon is initially added to the alkane chain subterminally at the C-2 position, with subsequent formation of
fatty acids.
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MATERIALS AND METHODS |
Source of organism and culture conditions.
The
alkane-degrading bacterial strain AK-01 was isolated from a
petroleum-contaminated estuarine sediment collected from the Arthur
Kill in New York (35). The medium used for maintaining AK-01
was the same as that described previously (35) except that
no yeast extract was added. Liquid alkanes and 1-alkenes used as the
substrates were sterilized separately by either autoclaving or
filtration (0.22 µm pore size) before use. Long-chain fatty acids (as
sodium salts) were prepared anoxically as concentrated stock solutions
and were autoclaved before being added (while still melted) to the
medium. Solid 1-alkanols were autoclaved together with the medium. In
most cases, subcultures of AK-01 grown on alkanes for about a month
were used as the inocula, which differed in size with the experiment
(see details for each experiment below). All cultures were incubated in
the dark without shaking.
Effect of alkanes on total cellular fatty acid composition.
Bottles containing 100 ml of medium were amended with 20 µl of
tetradecane, pentadecane, hexadecane, or heptadecane (99%; Sigma
Chemical Co., St. Louis, Mo.) and inoculated with cultures of AK-01
maintained on hexadecane (5%, vol/vol). For each alkane, two
experimental cultures and two sterile controls were set up. After
growing for about a month, cultures were subcultured again in medium
supplemented with the same alkane. After incubation for 38 to 51 days
to reach the stationary phase (growth rates differed depending on the
substrate), all cultures were sacrificed for extraction and analysis of
total cellular fatty acids.
Effect of other substrates on total cellular fatty acid
composition.
Bottles containing 100 ml of medium were amended with
the following substrates: 1-pentadecene (20 µl), 1-hexadecene (20 µl), 1-pentadecanol (20 mg), 1-hexadecanol (20 mg), pentadecanoate (0.5 mM), and hexadecanoate (0.5 mM). The media were then inoculated with cultures of AK-01 maintained on hexadecane (5%, vol/vol). For
each substrate, two experimental cultures and two autoclaved controls
were prepared. After incubation for 14 to 51 days to reach stationary
phase, the cultures were sacrificed for extraction and analysis of
total cellular fatty acids.
Degradation of
[1,2-13C2]hexadecane.
Cultures were
prepared by adding 20 µl of
[1,2-13C2]hexadecane (isotopic enrichment,
99%; Isotec Inc., Miamisburg, Ohio) to 100 ml of medium and
inoculating with a culture pregrown on pentadecane (5%, vol/vol). Two
experimental cultures and one sterile control were prepared. For the
sterile control, the inoculum was autoclaved before use. All cultures
were incubated in the dark without shaking for 47 days and sacrificed
for extraction and analysis of total cellular fatty acids.
Degradation of perdeuterated pentadecane.
Cultures were
prepared by adding 20 µl of perdeuterated pentadecane
(C15D32; isotopic enrichment, 98%; Cambridge
Isotope Laboratories, Inc., Andover, Mass.) to 100 ml of medium and
inoculating with a culture pregrown on hexadecane (5%, vol/vol).
Similar to the study described above, two experimental cultures and one
sterile control were prepared for the experiment, and the inoculum was autoclaved before use as the sterile control. All cultures were incubated in the dark without shaking for 39 to 47 days and then sacrificed for extraction and analysis of total cellular fatty acids.
Alkane degradation in the presence of
[13C]bicarbonate.
The mineral salt medium was
prepared with NaH13CO3 (isotopic enrichment,
99%; Cambridge Isotope Laboratories) instead of unlabeled sodium
bicarbonate for growth of the cultures in this experiment. Each bottle
containing 50 ml of medium was amended with 20 µl of pentadecane or
hexadecane and then inoculated with a hexadecane-grown culture (1%,
vol/vol; a small inoculum size was used to minimize [12C]bicarbonate contamination from the inoculum).
Duplicates of active cultures were prepared for each alkane. Cultures
were incubated for 45 to 67 days and then sacrificed for extraction and
analysis of total cellular fatty acids.
Extraction of total cellular fatty acids.
Each culture was
sacrificed by acidification with HCl (0.05 N final concentration), and
cells were collected by filtering them through a Teflon filter (0.5 µm pore size, 47 mm diameter; Gelman Sciences, Inc., Ann Arbor,
Mich.). The collected cells were transferred to a screw-capped glass
test tube with a Teflon-faced septum. A method to extract the total
cellular fatty acids from the cells was adapted from a protocol for
fatty acid analysis (Sherlock Microbial Identification System: MIDI,
Inc., Newark, Del.). To each tube with cells on the filter was added 2 ml of a NaOH-methanol solution (45 g of NaOH in 150 ml of methanol plus 150 ml of deionized water); the tubes were then capped and heated at
100°C for 30 min to release the cellular fatty acids by
saponification and cooled. Then 4 ml of an HCl-methanol solution (325 ml of 6 N HCl and 275 ml of methanol) was added to each tube, and the tubes were heated at 80°C for 10 min to esterify the fatty acids. When the tube contents had cooled down, 1.25 ml of hexane-methyl tert-butyl ether (1:1) was added to extract the fatty acid
methyl esters. The solvent extract was then washed with an aqueous NaOH solution (1.2%, wt/vol) to remove any unesterified fatty acids. Samples were stored in glass vials for subsequent analysis by gas
chromatography-mass spectrometry (GC-MS).
Analysis of fatty acid methyl esters.
Samples of fatty acid
methyl esters were initially analyzed by GC-flame ionization detection
(FID) with a gas chromatograph (model 5890, series II; Hewlett-Packard,
Wilmington, Del.) equipped with a flame ionization detector and a
DB-WAX column (30 m by 0.25 mm; film thickness, 0.25 µm; J&W
Scientific, Folsom, Calif.). The column temperature was initially set
at 50°C for 2 min, then increased to 140°C by 15°C per min and,
finally, to 220°C by 4°C per min. The injector and detector
temperatures were 250 and 300°C, respectively. The n-saturated and
monounsaturated fatty acids were identified by comparing their
retention times with those of commercially available authentic
standards (Sigma Chemical Co.).
Samples were also analyzed by GC-MS with a 5890 series II gas
chromatograph (Hewlett-Packard) equipped with a DB-5ms column (30 m by
0.25 mm; film thickness, 0.25 µm; J&W Scientific) and a 5971 series
mass-selective detector (Hewlett-Packard). The temperature program was
as follows: 60°C for 1 min, increased by 20°C per min to 200°C,
and then increased by 4°C per min to 280°C. The temperature for the
injector and detector was 280°C. Identities of most n-saturated and
unsaturated fatty acids were determined by comparing their retention
times and mass spectra with those of commercially available authentic
standards (Sigma Chemical Co.). Since no authentic standards were
available for them, the various methylated and isotope-labeled fatty
acids were identified by interpretation of their mass spectra based on
information reported in the literature (11, 16, 26, 28, 33).
Specific locations of 13C or deuterium atoms on the
isotope-labeled fatty acids were determined by examining the
diagnostically important ion fragments whose structural compositions
had been previously determined (10, 12, 26, 37a). Each fatty
acid in a sample was quantified based on its peak area in the total-ion
chromatogram and was expressed as a percentage of the total fatty acids
recovered. Preliminary analytical studies of a standard mixture of
fatty acid methyl esters on the GC-MS instrument showed that those with
chain lengths of C12 to C24 had similar
response ratios based on the total mass of each compound injected.
Nomenclature for fatty acids.
The fatty acid nomenclature
recommended by the IUPAC-IUB (20) was adopted in this study.
An n-saturated hexadecanoic acid is designated as 16:0, with the first
number representing the number of carbons in the acyl group and the
second number representing the number of double bonds present. The
branched fatty acid 2-methylhexadecanoic acid, for example, is
designated as 2-Me-16:0.
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RESULTS |
Effect of alkanes on total cellular fatty acid composition.
Figure 1 shows the fatty acid profiles of
strain AK-01 grown on pentadecane (Fig. 1a) and hexadecane (Fig. 1b) as
determined by GC-FID. The effect of the chain lengths of the alkane
substrates on the total cellular fatty acid composition is apparent.
When pentadecane was used as the growth substrate, the 13:0 and 15:0 fatty acids, both C odd, predominated over the C-even fatty acids. In
contrast, when hexadecane was used, the 14:0 and 16:0 fatty acids
became dominant while the C-odd fatty acids were virtually absent.
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FIG. 1.
Gas chromatograms (GC-FID) showing the fatty acid
profiles of strain AK-01 grown on pentadecane (a) and hexadecane (b).
The n-saturated fatty acids identified by their retention times are
annotated. C-odd fatty acids are underlined.
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The total cellular fatty acid composition of AK-01 upon growth on
different alkanes (C
14 to C
17) was further
studied in detail
by using GC-MS. As summarized in Fig.
2, when tetradecane (Fig.
2a) and
hexadecane (Fig.
2c), both C-even alkanes, were used as
growth
substrates, the cellular fatty acids of AK-01 were predominantly
C even
(comprising 89.3 and 97.1% of the total, respectively).
On the other
hand, when pentadecane (Fig.
2b) and heptadecane
(Fig.
2d) (both C odd)
were used, C-odd fatty acids were dominant
(65.6 and 91.1% of the
total, respectively). No abiotic formation
of these fatty acids was
observed for any of the sterile controls.
The carbon numbers of other
aliphatic compounds, such as 1-alkenes
(C
15 and
C
16), 1-alkanols (C
15 and C
16), and
fatty acids (C
15 and C
16), also had the same
effect on the fatty acid composition
of AK-01 when these compounds were
used as growth substrates (data
not shown). The results, therefore,
support the hypothesis that
alkanes (and the other aliphatic compounds)
are oxidized to fatty
acids by strain AK-01.
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FIG. 2.
Relative abundance of the total cellular fatty acids of
strain AK-01 grown on tetradecane (a), pentadecane (b), hexadecane (c),
and heptadecane (d). Only those which can be identified and comprise
more than 1% of the total are shown. C-even fatty acids are
represented by black bars, and C-odd fatty acids are represented by
white bars. The 2-methyl, 4-methyl, and 6-methyl fatty acids are
denoted by the numbers 2, 4, and 6, respectively. (The 2-Me-17:0 fatty
acid is also shown, though it constitutes only 0.7% of the total.) No
fatty acid was observed in the sterile controls.
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It should be noted that some monomethylated fatty acids with methyl
groups located at the C-2, C-4, or C-6 position (as indicated
in Fig.
2) were only found when the organism is grown on alkanes,
not found
when it was grown on 1-alkenes, 1-alkanols, or fatty
acids. In
addition, the chain lengths of these monomethylated
fatty acids also
specifically correlated with those of the alkane
substrates. As shown
in Fig.
2a, the 2-Me-14:0, 4-Me-16:0, and
6-Me-18:0 fatty acids were
found only when tetradecane (C
14) was
used as the
substrate. The 2-Me-15:0, 4-Me-17:0, and 6-Me-19:0
fatty acids were
found only when pentadecane (C
15) was used as
the substrate
(Fig.
2b). The same pattern was also observed in
cultures grown on
hexadecane or heptadecane (Fig.
2c and d). Furthermore,
these
monomethylated fatty acids were uniquely found in strain
AK-01 only
when it was grown on alkanes, and they have not been
reported to occur
in other sulfate-reducing bacteria (
36).
C
10-methylated
fatty acids, however, were found in strain
AK-01 when it was grown
on the other aliphatic substrates as well as
alkanes. In addition,
10-methylated fatty acids can also be found in
other sulfate-reducing
bacteria incapable of alkane degradation, and
thus they are not
unique to strain AK-01 (
36).
The fact that these 2-, 4-, and 6-methylated fatty acids were only
observed when AK-01 was grown on alkanes is a strong indication
that
their formation is specifically related to the initial reactions
of
anaerobic oxidation of alkanes. Conversely, the absence of
these
methylated fatty acids when AK-01 is grown on 1-alkenes
and 1-alkanols
also suggests that the latter two classes of compounds
are probably not
intermediates in anaerobic alkane
metabolism.
Analyses of [1,2-13C2]hexadecane-derived
fatty acids.
Mass spectral analyses of the 2-, 4-, and
6-methylated fatty acids recovered from cultures of AK-01 grown on
[1,2-13C2]hexadecane confirmed that these
fatty acids were derived from the alkane. Figure
3 shows the mass spectra of the methyl
esters of the unlabeled (Fig. 3a) and 13C-labeled (Fig. 3b)
2-Me-16:0 fatty acids recovered from AK-01 cultures grown on unlabeled
hexadecane and [1,2-13C2]hexadecane,
respectively. As shown in Fig. 3a, the molecular ion peak for the
unlabeled 2-Me-16:0 fatty acid appears at m/z = 284. The molecular ion peak for the same fatty acid from the [1,2-13C2]hexadecane-grown cultures is
shifted up by 2 mass units to m/z = 286 (Fig. 3b),
reflecting the presence of two 13C atoms in the molecule.
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FIG. 3.
Mass spectra of the methyl esters of the unlabeled (a)
and 13C-labeled (b) 2-Me-16:0 fatty acids recovered from
cultures of strain AK-01 grown on unlabeled hexadecane and
[1,2-13C2]hexadecane, respectively. Chemical
structures of the fatty acids represented by the mass spectra are shown
as insets. The key diagnostic ion peaks are annotated with their
m/z values. Structural compositions of the ion fragments
represented by the annotated peaks are delineated. Intersection with a
dotted line indicates a point of bond cleavage, and the ion fragment
formed contains only the part of the molecule on the left side of the
dotted line. (Note: the mass spectrum for the 13C-labeled
2-Me-16:0 fatty acid indicates the coexistence of two isotopmers.)
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A detailed examination of the mass spectra shows that the initial
attack can occur at either the labeled or unlabeled end
of the
[1,2-
13C
2]hexadecane. As depicted in Fig.
3a,
the ion fragment represented
by the ion peak at
m/z = 88 includes C-1, C-2, and the 2-methyl
group of the unlabeled 2-Me-16:0
fatty acid (along with the ester-linked
methyl group). This fragment is
formed by a process, known as
the McLafferty rearrangement, which
specifically involves the
migration of a hydrogen from C-4 to the
carbonyl oxygen and the
cleavage of the C-2-C-3 bond (
26,
33). Furthermore, the ion
fragment at
m/z = 143 includes C-1 to C-6 (and the 2-methyl group)
of the unlabeled fatty
acid and is formed by the cleavage of the
C-6-C-7 bond (
12,
26,
33).
On the mass spectrum for the
13C-labeled 2-Me-16:0 fatty
acid (Fig.
3b), peaks of comparable intensity appear at
m/z = 88 and
90. Thus, this ion fragment, which
includes C-1, C-2, and the
2-methyl group of the fatty acid, occurs in
two forms that are
present in comparable abundance but that contain
different numbers
of
13C atoms. The one at
m/z = 90 contains two
13C atoms, while the
one at
m/z = 88 contains none at all. It appears,
therefore, that the
13C-labeled 2-Me-16:0 fatty acid occurs
as two isotopmers of comparable
abundance. One isotopmer contains two
13C atoms among the C-1, C-2, and the 2-methyl group
carbons (isotopmer
A in Fig.
3b), while the other has the two labels
located at the
other end of the molecule (isotopmer B). Isotopmers are
versions
of the same molecule with different specific isotopic
labelling
patterns (
33a).
Further analysis of the mass spectrum of the
13C-labeled
2-Me-16:0 fatty acid showed that isotopmer B contained two
13C atoms between C-7 and C-16. As shown in Fig.
3b, two
peaks of
comparable intensity appear at
m/z = 143 and
145, indicating again
the occurrence of two isotopmers. One (isotopmer
A) contains two
13C atoms between C-1 and C-6 (thus,
m/z = 145), while the other
(isotopmer B) has the two
labels located between C-7 and C-16
(thus,
m/z = 143).
These two labels in isotopmer B are most likely
located at C-15 and
C-16, their original locations on the
[1,2-
13C
2]hexadecane. The presence of two
13C atoms at either end of the fatty acid, therefore,
indicates
that the initial attack occurs with the same likelihood at
the
labeled and the unlabeled termini of the
hexadecane.
In addition, further examination of the mass spectra indicated that the
two
13C atoms in isotopmer A of the 2-Me-16:0 fatty acid
are located
at C-2 and the 2-methyl group. As illustrated in Fig.
4a, the
ion peak at
m/z = 241 for the unlabeled fatty acid (indicated
in the mass spectrum in
Fig.
3a) is formed by the elimination
of the segment of the molecule
containing C-2, C-3, and the 2-methyl
group (and an additional
hydrogen, yielding a combined mass of
43) followed by the rearrangement
of the remaining parts. This
fragmentation mechanism is documented in
the literature (
12,
33,
37a). The corresponding ion peak in
the mass spectrum of
the
13C-labeled fatty acid appears as
a doublet of peaks of comparable
intensity at
m/z = 241 and 243 (Fig.
3b), indicating that isotopmer
A contains two
13C atoms in the eliminated segment (Fig.
4b) while
isotopmer B
does not (Fig.
4c). The results show that the two
13C atoms in isotopmer A are located at C-2, C-3, and/or
the 2-methyl
group, not at C-1 or elsewhere (Fig.
4b). Furthermore, as
shown
earlier by examination of the doublet at
m/z = 88 and 90, none
of the two
13C carbons on isotopmer A is
located at C-3. Thus, they are located
at C-2 and the 2-methyl group of
isotopmer A.
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FIG. 4.
Ion fragments represented by the peaks at
m/z = 241 for the unlabeled 2-Me-16:0 fatty acid methyl
ester (a) (see Fig. 3a) and m/z = 241 (b) and
m/z = 243 (c) for the 13C-labeled 2-Me-16:0
fatty acid methyl ester (see Fig. 3b). These ion fragments are formed
by the elimination of the bracketed segment of the molecule and a
hydrogen, followed by the rearrangement of the remaining parts
(12, 33, 37a).
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Based on the results described above, we conclude that the 2-methyl
group of the 2-Me-16:0 fatty acid is the original terminal
carbon of
the [1,2-
13C
2]hexadecane. This suggests that
exogenous carbon is initially
added onto the
[1,2-
13C
2]hexadecane subterminally at the
C-2 position such that the
original terminal carbon of the alkane then
becomes the methyl
group of the resulting 2-Me-16:0 fatty
acid.
Mass spectral data on the 4-Me-18:0 fatty acid from the
[1,2-
13C
2]hexadecane-grown cultures also
indicate that the 4-methyl group
is the original terminal carbon of the
alkane. Figure
5 shows
the mass spectra
of the unlabeled (Fig.
5a) and
13C-labeled (Fig.
5b)
4-Me-18:0 fatty acid recovered from cultures
grown on unlabeled
hexadecane and [1,2-
13C
2]hexadecane,
respectively. The presence of two
13C atoms in the latter
fatty acid is indicated by the molecular
ion at
m/z = 314 (Fig.
5b).
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FIG. 5.
Mass spectra of the methyl esters of the unlabeled (a)
and 13C-labeled (b) 4-Me-18:0 fatty acids recovered from
cultures of strain AK-01 grown on unlabeled hexadecane and
[1,2-13C2]hexadecane, respectively. Chemical
structures of the fatty acids represented by the mass spectra are shown
as insets. (Note: the mass spectrum for the 13C-labeled
4-Me-18:0 fatty acid indicates the coexistence of two isotopmers.)
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The
13C-labeled 4-Me-18:0 fatty acid also occurs as two
isotopmers, one with the two
13C atoms located at C-4 and
the 4-methyl group. As shown in Fig.
5a, the ion fragment at
m/z = 87 is formed by cleavage of the
C-3-C-4 bond and
includes C-1 to C-3 of the unlabeled 4-Me-18:0
fatty acid. In addition,
the ion fragment at
m/z = 115 is formed
by cleavage of
the C-4-C-5 bond and includes C-1 to C-4 and the
4-methyl group of the
molecule (
12,
26,
33). The difference
between the
m/z values of these two ion peaks, which amounts to
28 mass
units, represents the combined mass of C-4 plus the attached
hydrogen
atom and the 4-methyl group [---CH(CH
3)---].
On the mass spectrum for the
13C-labeled 4-Me-18:0 fatty
acid (Fig.
5b), the ion peak corresponding to that at
m/z = 87 for
the unlabeled fatty acid (Fig.
5a) remains
the same, indicating
the absence of
13C label at C-1 to
C-3. The peak corresponding to
m/z = 115, however,
appears as a doublet of peaks at
m/z = 115 and 117 of
comparable
intensity. Thus, the
13C-labeled 4-Me-18:0 fatty
acid occurs as two isotopmers of comparable
abundance, one labeled with
two
13C atoms, at C-4 and the 4-methyl group (isotopmer A
in Fig.
5b),
and the other with no isotope label on these carbons. The
two
13C atoms in the latter case are most likely located at
C-17 and
C-18 (isotopmer B in Fig.
5b). These results shows that the
4-methyl
group is the original terminal carbon of the
[1,2-
13C
2]hexadecane and further
underscore the point that exogenous
carbon is added onto the alkane
subterminally at the C-2 position.
It also appears that the 4-Me-18:0
fatty acid is formed after
elongation of the 2-Me-16:0 fatty acid by
two carbon units through
fatty acid chain elongation reactions
(
31).
Table
1 summarize the characteristics of
the 2-, 4-, and 6-methylated fatty acids derived from the
[1,2-
13C
2]hexadecane. For each fatty
acid, the positions of the
13C atoms on each isotopmer and
the relative abundance of the two
different isotopmers are shown. None
of these
13C-labeled fatty acids was found in the sterile
control. Like the
2-Me-16:0 and 4-Me-18:0 fatty acids, the
13C-labeled 6-Me-20:0 fatty acid contains two
13C atoms, at C-6 and the 6-methyl group, in one isotopmer
(mass
spectrum not shown). Thus, it is a result of a similar carbon
addition mechanism followed by chain elongation. Other C-even
fatty
acids (14:0, 10-Me-14:0, 16:0, and 10-Me-16:0), which are
also
13C labeled, appear to be formed after further metabolism
of the
2-Me-16:0 fatty acid (data not shown).
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TABLE 1.
Characteristics of the 13C-labeled 2-, 4-, and 6-methylated fatty acids recovered from cultures of strain
AK-01 grown on [1,2-13C2]hexadecane
|
|
Analyses of perdeuterated pentadecane-derived fatty acids.
Mass spectral analyses of the deuterated fatty acids also indicate that
exogenous carbon is added onto the alkane chain subterminally to form a
monomethylated fatty acid. Figure 6 shows
the mass spectra of the methyl esters of the unlabeled (Fig. 6a) and
deuterated (Fig. 6b) 2-Me-15:0 fatty acid recovered from cultures grown
on unlabeled and perdeuterated pentadecane, respectively. As shown in
Fig. 6a, the ion fragment, m/z = 88, of the unlabeled
2-Me-15:0 fatty acid is formed by the McLafferty rearrangement as
described earlier (26, 33). The corresponding peak for the
deuterated fatty acid (Fig. 6b) is shifted up by 4 mass units
(m/z = 92), indicating the presence of four deuterium
atoms in the ion fragment. Since the methanol used for the fatty acid
derivatization was not deuterated and one deuterium would have been
contributed by the McLafferty rearrangement (12), we deduce
that three deuterium atoms are located at C-2 and the 2-methyl group.
Similar to the study with the
[1,2-13C2]hexadecane, the results here
indicate that the 2-methyl group of the deuterated 2-Me-15:0 fatty acid
is the original terminal carbon of the perdeuterated pentadecane. Thus,
the 2-Me-15:0 fatty acid is the result of subterminal carbon addition
to the perdeuterated pentadecane at the C-2 position.
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|
FIG. 6.
Mass spectra of the methyl esters of the unlabeled (a)
and deuterated (b) 2-Me-15:0 fatty acids recovered from cultures of
strain AK-01 grown on unlabeled and perdeuterated pentadecane,
respectively. Chemical structures of the fatty acids represented by the
mass spectra are shown as insets. Interpretation of the mass spectra
shows that there are three deuterium atoms, located on C-2 and the
2-methyl group of the deuterated 2-Me-15:0 fatty acid. The locations of
all three deuterium atoms on the 2-methyl group as shown in Fig. 6b
represent one possible arrangement.
|
|
Mass spectral analyses of the deuterated 4-Me-17:0 and 6-Me-19:0 fatty
acids from cultures grown on perdeuterated pentadecane
also indicated
the occurrence of subterminal carbon addition.
As shown in Table
2, the 2-, 4-, and 6-methylated fatty
acids
all contain three deuterium atoms, located on their methyl groups
and the adjacent carbons, indicating that the methyl groups also
contain the original terminal carbon of the perdeuterated pentadecane
and that exogenous carbon is added to the deuterated pentadecane
subterminally at C-2. This thus shows that the deuterated 4-Me-17:0
and
6-Me-19:0 fatty acids are formed by chain elongation of the
2-Me-15:0
fatty acid. Deuterium atoms are also found on the carbons
between the
carboxyl groups and methyl groups of the 4-Me-17:0
and 6-Me-19:0 fatty
acids (Table
2). This may be effected by
the initial carbon addition
reaction in that the carbon compound
being added to the perdeuterated
pentadecane is also directly
derived from the labeled alkane and thus
still carries deuterium
atoms. Other C-odd deuterated fatty acids,
namely 15:0 and 10-Me-15:0,
are also recovered and are likely formed by
further metabolism
of the 2-Me-15:0 fatty acid. The lack of deuterium
atoms in the
10-methyl group of the deuterated 10-Me-15:0 (data not
shown)
indicates that this C-10 methyl group is exogenously added, in
contrast to those of the 2-, 4-, and 6-methylated fatty acids.
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|
TABLE 2.
Characteristics of the deuterated, 2-, 4-, and
6-methylated fatty acids recovered from cultures of strain AK-01
grown on perdeuterated pentadecane
|
|
Fatty acids formed in the presence of
[13C]bicarbonate.
The 2-methylated fatty acids
recovered from cultures of AK-01 grown on unlabeled pentadecane and
hexadecane in the presence of [13C]bicarbonate were
characterized by mass spectrometry. The mass spectra of the 2-Me-15:0
and 2-Me-16:0 fatty acids formed in the presence of
[13C]bicarbonate appeared the same as those formed in the
presence of unlabeled bicarbonate. The results indicate that the
carboxyl groups of the 2-methylated fatty acids are not 13C
labeled. Hence, in AK-01, the carbon addition at C-2 of the alkane
chain does not appear to occur through the addition of an inorganic
carboxyl group.
| |
DISCUSSION |
The alkane-degrading strain AK-01 was isolated from
petroleum-contaminated estuarine sediments. It grows anaerobically on alkanes (C13 to C18) and is able to oxidize
them to carbon dioxide coupled to sulfate reduction (35). In
the present study, the mechanism for anaerobic alkane degradation by
the strain was determined. Although 1-alkene and 1-alkanol have been
suggested as intermediates (29) and strain AK-01 is able to
utilize both for growth (35), neither compound was found in
the alkane-grown cultures in our experiments. That they are unlikely to
be intermediates is also supported by data from studies of strain Hxd3
(1, 3).
Although no free fatty acid was extractable from whole cultures of
AK-01 grown on alkanes, the impact of the alkane substrates' carbon
numbers on the total cellular fatty acid composition (Fig. 2) is highly
suggestive of alkanes being oxidized to fatty acids by the organism.
This has been observed with the other alkane-utilizing sulfate reducers
(3, 14) and also for microorganisms capable of aerobic
alkane utilization (13, 21). The fatty acids formed after
alkane oxidation can be directly incorporated into the cellular lipids
of the alkane-degrading organisms (5, 18, 22).
The recovery of deuterated and 13C-labeled fatty acids in
cultures of AK-01 grown on the perdeuterated and
13C-labeled alkanes, respectively, clearly confirms that
alkanes are anaerobically oxidized to fatty acids. In addition, the
carbon numbers of the labeled fatty acids also correlate with those of the alkane substrates. When
[1,2-13C2]hexadecane was used as a growth
substrate, the recovered 13C-labeled fatty acids were all C
even (Table 1). When perdeuterated pentadecane was used, the recovered
deuterated fatty acids were all C odd (Table 2). The results show that
the fatty acids derived from the alkanes can be 
-oxidized and
mineralized to CO2 (24, 35) or further
transformed through chain elongation and C-10 methylation
(31) by AK-01.
Evidence presented here for a proposed mechanism of alkane oxidation is
derived from mass spectral analyses of the resulting stable-isotope-labeled fatty acids. The mass spectra of the methyl esters of many unlabeled and deuterated fatty acids have been reported
(11, 16, 26, 28, 33). The compositions and fragmentation
mechanisms of various ion fragments, which contain different parts of
the fatty acid molecule, are also well studied (10, 12, 26).
Based on this information, many fatty acids can be identified and the
locations of 13C and deuterium atoms on the isotope-labeled
fatty acids can be determined.
Three lines of evidence support a mechanism by which exogenous carbon
is added to an alkane subterminally as an initial step in anaerobic
alkane metabolism. First, the 2-Me-16:0, 4-Me-18:0, and 6-Me-20 fatty
acids derived from the [1,2-13C2]hexadecane
each contains two 13C atoms, located at the methyl group
and the adjacent carbon (Table 1). The results indicate that exogenous
carbon is added subterminally to the hexadecane at the C-2 position,
such that the terminal carbon of the alkane becomes the 2-, 4-, and
6-methyl groups of the fatty acids formed subsequently.
Second, the 2-Me-15:0, 4-Me-17:0, and 6-Me-19 fatty acids derived from
the perdeuterated pentadecane each contains three deuterium atoms,
located on the methyl group and the adjacent carbon (Table 2). This
also supports the same mechanism, namely subterminal carbon addition to
the perdeuterated pentadecane at C-2, resulting in the original
terminal carbon of the alkane forming the 2-, 4-, and 6-methyl groups
of these fatty acids.
Third, the 2-, 4-, and 6-methylated fatty acids are found only in
cultures grown on alkanes, and their chain lengths always correlate
with those of the alkane substrates (Fig. 2). These uncommon
monomethylated fatty acids are not observed when AK-01 is grown on
other aliphatic substrates (1-alkenes, 1-alkanols, and fatty acids),
and they are not observed in other sulfate-reducing bacteria
(36). Therefore, these fatty acids are formed specifically by the initial reactions of alkane oxidation. Furthermore, it appears
that the 2-methylated fatty acids are initially formed from the alkanes
and can be subsequently elongated by two carbon units to form the
4-methylated and 6-methylated fatty acids through the fatty acid
elongation reactions (Tables 1 and 2) (31). The nature of
the carbon compound being added onto the alkane chain, however, remains
unknown at this time. The absence of the 2-, 4-, and 6-methylated fatty
acids in AK-01 cultures grown on 1-alkenes also indicates that carbon
addition is not mediated by the formation of a terminal double bond in
the alkane.
Although carboxylation has been demonstrated as an initial reaction of
aromatic hydrocarbon metabolism under anaerobic conditions (30,
39), results from our experiment with
[13C]bicarbonate show that the subterminal carbon
addition is probably not due to a direct carboxylation. On the other
hand, it has been demonstrated that toluene is anaerobically
metabolized by addition of fumarate to form benzyl succinate and then
benzoyl coenzyme A (4). Hence, we cannot discount the
possibility that a compound with more than one carbon is added to an
alkane to form the first intermediate, which is then transformed into a
2-methylated fatty acid.
The anaerobic attack on alkanes by AK-01 involves carbon addition at
the C-2 position. Such selectivity for the subterminal carbon may be
caused by the conformational structure of the degrading enzyme. Studies
with model systems support the hypothesis that the terminal carbons of
alkanes are selectively hydroxylated by cytochrome P-450 because of
steric hindrance at its active site (23). The degrading
enzyme of AK-01 may have a less sterically hindered active site, such
that the subterminal carbon at C-2 can be attacked. Furthermore, as a
secondary carbon, its C---H bond is weaker than that of a terminal,
primary carbon (bond dissociation energy, 94.5 versus 98 kcal/mol for
propane, for example) (15) and thus should be more readily broken.
Based on the collective observations in this study, a proposed pathway
for anaerobic oxidation of alkanes by strain AK-01 is summarized in
Fig. 7. An alkane (A) is initially
attacked subterminally by the addition of a carbon compound at the C-2
position to form a 2-methylated fatty acid (B). This fatty acid then
undergoes -oxidation (24, 25, 34) to form an n-saturated
fatty acid (C) which can be further -oxidized and eventually
mineralized to CO2 (35). In addition, the
initially formed 2-methylated fatty acid (B) can also be elongated to
form the 4-methylated (D) and 6-methylated (E) fatty acids.
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|
FIG. 7.
Proposed pathway for anaerobic alkane metabolism by
strain AK-01. (The original alkane atoms are boldfaced, and the major
pathway is indicated by bold arrows.)
|
|
In summary, we provide direct evidence that alkanes are oxidized to
fatty acids by strain AK-01 under strictly anaerobic conditions. Subterminal addition of a carbon(s) to the hydrocarbon chain as an
initial reaction in anaerobic alkane degradation is also demonstrated through a detailed characterization of these fatty acid metabolites. This carbon addition reaction represents a novel mechanism by which
alkanes can be activated without oxygen, in contrast to the well-known
hydroxylation reaction mediated by monooxygenases in aerobic organisms.
| |
ACKNOWLEDGMENTS |
We thank Max Häggblom, Xiaoming Zhang, Mike Logan, Craig
Phelps, and Alfred Boyle for technical advice and editorial help, Ingeborg Bossert and Andreas Naef for translation of German literature, and Maria Rivera and Brian Donovan for technical assistance.
This work was supported in part by the Office of Naval Research and the
Defense Advanced Research Project Agency.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Biotechnology
Center for Agriculture and the Environment, Foran Hall, Cook College, Rutgers, The State University of New Jersey, 59 Dudley Rd., New Brunswick, NJ 08901-8520. Phone: (732) 932-8165, ext. 312. Fax: (732)
932-0312. E-mail: lyoung{at}aesop.rutgers.edu.
Present address: Department of Biology, The Chinese University of
Hong Kong, Shatin, N.T., Hong Kong SAR, People's Republic of China.
| |
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Top
Abstract
Introduction
