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Appl Environ Microbiol, April 1998, p. 1447-1453, Vol. 64, No. 4
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Use of 13C Nuclear Magnetic Resonance
To Assess Fossil Fuel Biodegradation: Fate of
[1-13C]Acenaphthene in Creosote Polycyclic Aromatic
Compound Mixtures Degraded by Bacteria
Sergey A.
Selifonov,1,*
Peter J.
Chapman,2
Simon B.
Akkerman,1
Jerome E.
Gurst,3
Jacqueline M.
Bortiatynski,4
Mark A.
Nanny,4 and
Patrick G.
Hatcher4
Department of Biochemistry and Institute for
Advanced Studies in Biological Process Technology, University of
Minnesota, Gortner Laboratory, St. Paul, Minnesota
551081;
Gulf Ecology Division, National
Health and Environmental Effects Research Laboratory, U.S.
Environmental Protection Agency, Gulf Breeze, Florida
325612;
Department of Chemistry,
University of West Florida, Pensacola, Florida
325143; and
Fuel Science Program,
The Pennsylvania State University, University Park, Pennsylvania
168024
Received 21 August 1997/Accepted 30 January 1998
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ABSTRACT |
[1-13C]acenaphthene, a tracer compound with a nuclear
magnetic resonance (NMR)-active nucleus at the C-1 position, has been employed in conjunction with a standard broad-band-decoupled
13C-NMR spectroscopy technique to study the biodegradation
of acenaphthene by various bacterial cultures degrading aromatic
hydrocarbons of creosote. Site-specific labeling at the benzylic
position of acenaphthene allows 13C-NMR detection of
chemical changes due to initial oxidations catalyzed by bacterial
enzymes of aromatic hydrocarbon catabolism. Biodegradation of
[1-13C]acenaphthene in the presence of naphthalene or
creosote polycyclic aromatic compounds (PACs) was examined with an
undefined mixed bacterial culture (established by enrichment on
creosote PACs) and with isolates of individual naphthalene- and
phenanthrene-degrading strains from this culture. From
13C-NMR spectra of extractable materials obtained in time
course biodegradation experiments under optimized conditions, a number of signals were assigned to accumulated products such as
1-acenaphthenol, 1-acenaphthenone, acenaphthene-1,2-diol and
naphthalene 1,8-dicarboxylic acid, formed by benzylic oxidation of
acenaphthene and subsequent reactions. Limited degradation of
acenaphthene could be attributed to its oxidation by naphthalene
1,2-dioxygenase or related dioxygenases, indicative of certain
limitations of the undefined mixed culture with respect to acenaphthene
catabolism. Coinoculation of the mixed culture with cells of
acenaphthene-grown strain Pseudomonas sp. strain A2279
mitigated the accumulation of partial transformation products and
resulted in more complete degradation of acenaphthene. This study
demonstrates the value of the stable isotope labeling approach and its
ability to reveal incomplete mineralization even when as little as 2 to
3% of the substrate is incompletely oxidized, yielding products of
partial transformation. The approach outlined may prove useful in
assessing bioremediation performance.
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INTRODUCTION |
Microbial degradation of polycyclic
aromatic compounds (PACs) is increasingly being considered for
bioremediation applications and has been proposed as an attractive
approach to remediation technologies dealing with fossil fuel wastes
(10, 11). However, biodegradation of PACs of creosote and of
related hydrocarbons of petroleum by defined and undefined mixed
bacterial cultures may result in elevated rates of formation and even
accumulation of organic end products (3, 5, 20). Some of
these products are toxic to certain test organisms (1, 3,
20). Such information may be relevant in determining, for
example, why little change in toxicity is observed when creosote
undergoes biodegradation in groundwater (11). Therefore,
chemical and toxicological characterization of any biodegradation
process is required before the method is applied.
A realistic assessment of biodegradation efficacy requires delineation
of a pollutant's fate and effects beyond its depletion, as revealed by
sensitive and exact analytical methods. For complex mixtures of
chemicals, such as those found in coal- and petroleum-derived materials, this is not so readily accomplished. Assessment of efficacy
cannot be based solely on rates of mineralization inferred from data
obtained by trapping CO2 released after the addition of a
radioactively labeled tracer compound. Although this method is a
convenient laboratory indicator of limited aspects of mineralization, it fails to provide information about the presence, nature, and distribution of organic end products or their interactions with soil
and sedimentary matter.
13C-nuclear magnetic resonance (NMR) spectroscopy, combined
with 13C labeling, however, offers an approach whereby
details of the chemical structures of individual components of PAC
mixtures undergoing biodegradation can be investigated. With the
13C-labeled tracers available, 13C-NMR has been
successfully applied to an investigation of the interaction of
2,4-dichlorophenol with humic matrixes (8) and to a study of
the alteration of jet fuels undergoing thermal stress (9).
For a more complete study of its utility, various specifically 13C-labeled constituents of fossil fuels are required for
evaluation of this technique in assessing the biodegradation of coal-
and petroleum-derived wastes.
The applicability of this approach to the biodegradation of hydrocarbon
mixtures is examined in the present study by using synthetic
[1-13C]acenaphthene (99% 13C) introduced as
a tracer compound into creosote PAC mixtures degraded by different
undefined, mixed, and axenic bacterial cultures. The initial choice of
acenaphthene labeled with 13C at a benzylic carbon as a
tracer is based on the abundance of acenaphthene in creosote and the
straightforward route of its synthesis. It also represents an extension
of published work on acenaphthene oxidation catalyzed by naphthalene
dioxygenase (16), biotransformation reactions catalyzed by
bacteria and fungi (12, 14), and catabolism of acenaphthene
by certain soil bacteria (19), with the consequent
availability of suitable acenaphthene-utilizing microbial cultures.
(Preliminary accounts of aspects of this work have appeared elsewhere
[2, 17, 18].)
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MATERIALS AND METHODS |
Chemicals.
PACs were purified from creosote P2 (Aristech
Chemical Corp., Clairton, Pa.) by a modification of a previously
described procedure (6, 13). Characterization of the
composition of the PAC fraction and quantitative analysis of the
principal PAC components, using as a reference a standard coal tar
material, SRM1597 (National Institute of Standards and Technology,
Gaithersburg, Md.) (21), were reported previously
(6). Authentic acenaphthenone and cis- and
trans-acenaphthene-1,2-diols were available from a previous study (16). All the commercial chemicals were of the highest purity available.
[1-13C]Acenaphthene was obtained by a four-step synthesis
K13CN (670 mg, 99% 13C; Aldrich Chemical Co.,
Milwaukee, Wis.) was added to a stirred solution of 2.1 g of
1-(chloromethyl)naphthalene in 20 ml of aqueous ethanol (70%, vol/vol)
to give 1'-naphthyl-[1-13C]acetonitrile (not isolated).
After the mixture was refluxed for 1 h, 20 ml of a 30% (wt/vol)
solution of NaOH in water was added. Refluxing was continued until
evolution of ammonia ceased (approximately 6 h). The reaction
mixture was cooled and washed three times with 50 ml of diethyl ether.
The aqueous layer was separated and acidified to pH 2.0 to 2.5 by the
addition of concentrated HCl. Precipitated
1'-naphthyl-[1-13C]acetic acid was extracted into ethyl
acetate and recrystallized from diethyl ether (1.62 g, 92% yield,
based on K13CN). Intramolecular acylation of
1'-naphthyl-[1-13C]acetic acid (0.8 g) was performed by
addition of 30 ml of hot 85% polyphosphoric acid (110 to 130°C) with
vigorous stirring for 4 to 5 min. The reaction was stopped by the rapid
addition of ice-water (50 ml). The solution was adjusted to pH 8 to 9 by the addition of NaOH, and crude [1-13C]acenaphthenone
was extracted into diethyl ether. [1-13C]acenaphthenone
was purified by sublimation to remove colored dimeric products. When
performed repeatedly, the cyclization reaction gave
[1-13C]acenaphthenone with an overall yield in the range
of 42 to 63%. Reaction yields were strongly dependent on the
temperature of the polyphosphoric acid, the incubation time, and the
stirring regimen.
[1-
13C]Acenaphthenone was converted to
[1-
13C]acenaphthene by Clemmensen reduction.
[1-
13C]acenaphthenone (400 mg) was added to 30 ml of 10%
aqueous HCl
mixed with 0.05% (vol/vol) toluene and 3 g of
amalgamated zinc
wool. The mixture was refluxed for 4 h.
[1-
13C]acenaphthene was recovered from the reaction
mixture by extraction
into
n-hexane and was purified by
sublimation to yield 337 mg
(92% yield). The structures of the product
and precursors were
confirmed by mass spectrometry and NMR
spectroscopy; the spectra
were compared to those of authentic unlabeled
materials. The sublimed
[1-
13C]acenaphthene (99%
13C-1) was of 98% purity, as determined by gas
chromatography (GC),
with a single impurity identified as
[1-
13C]acenaphthylene (2%).
Microorganisms.
An undefined mixed culture of bacteria
(CREOMIX) was obtained from creosote-contaminated soil from the
American Creosote Works site in Pensacola, Fla. (3). The
creosote PAC fraction, as the sole carbon and energy source in a
mineral salts medium (7), was used to establish the
enrichment culture. The culture was maintained at 20 to 25°C and
transferred biweekly. This culture has been previously shown to
completely deplete all constituents with three or fewer rings in the
PAC mixture within 14 days of incubation. No significant degradation of
compounds with four or more rings was observed in this culture, and no
further depletion of PACs occurred during periods of incubation
extending beyond 2 weeks. Complex mixtures of low-molecular-weight
aromatic compounds were accumulated by this culture as end products of
the PAC degradation (3). The CREOMIX culture was maintained
for more than 3 years without loss of performance before it was used in
this study.
Two individual strains, BR and BC, were isolated from the CREOMIX
culture and tentatively identified as
Pseudomonas spp. Both
of these strains grew with either naphthalene or phenanthrene,
but not
on acenaphthene, as the sole carbon source and were used
in this study
to determine their contribution in the accumulation
of biodegradation
products from acenaphthene.
Attempts to isolate from the CREOMIX culture any individual strains of
bacteria able to utilize acenaphthene as the sole carbon
and energy
source were not successful. The acenaphthene-catabolizing
Pseudomonas sp. strain A2279 used in this study was obtained
from
the same creosote-contaminated soil by enrichments with
acenaphthene
as the sole carbon and energy source. This organism also
grows
with acenaphthylene or naphthalene as the sole carbon and energy
source. Acenaphthene and acenaphthylene are degraded by this organism
via naphthalene-1,8-dicarboxylic acid and
2-hydroxybenzene-1,3-dicarboxylic
acid (
15).
Biodegradation experiments.
All biodegradation experiments
involving the use of [13C]acenaphthene and growth of all
inoculum cultures were performed in 125-ml Erlenmeyer flasks with 25 ml
of mineral salts medium (7) and with the pH adjusted to 7.2. Aliquots of stock solutions of the test compounds in methylene chloride
were added to empty sterile flasks. Sterilized medium was added to the
flasks after evaporation of the solvent (usually after 2 to 4 h).
Uninoculated flasks were used as controls for extraction efficiency,
losses by volatilization, and abiotic degradation.
The individual naphthalene-degrading strains BR and BC were grown in 25 ml of the liquid mineral salt medium with 1 g of naphthalene
per
liter until the late exponential phase (approximately 36 h).
The
acenaphthene-degrading strain A2279 was grown similarly with
1 g
of acenaphthene per liter. Aliquots (1 ml) of the cultures
were used to
inoculate a series of replicate flasks, which contained
either (i) a
mixture of 10 mg of [1-
13C]acenaphthene (previously
diluted with unlabeled acenaphthene
to give 20%
13C) and
10 mg of naphthalene or (ii) a mixture of 25 mg of creosote
PACs and 1 mg of [
13C]acenaphthene (99%
13C).
To examine the action of the undefined CREOMIX mixed culture on
acenaphthene, the culture was first grown on 1 g of creosote
PACs
per liter for 2 weeks. Aliquots (1 ml) of this culture were
then used
to inoculate replicate flasks containing a mixture of
25 mg of creosote
PACs and 1 mg of [
13C]acenaphthene (99%
13C). A separate series of replicate flasks coinoculated
with 1
ml of the CREOMIX culture and 1 ml of an acenaphthene-grown
culture
of strain A2279 was used to elucidate the effects of the latter
organisms.
The flasks were incubated at 25°C on a rotary shaker with shaking at
200 rpm in the dark. In time course experiments, flasks
were collected
and their contents were acidified to pH 2 to 3
by the addition of 5 N
HCl. The entire flask contents were extracted
three times with 10 ml of
methylene chloride. The extracts were
dried over anhydrous sodium
sulfate and concentrated under a stream
of N
2 to 25 ml in
Kuderna-Danish evaporative tubes without application
of heat or reduced
pressure. Aliquots (1 ml) were taken for GC
analysis with flame
ionization detection (FID). The remainder
of each extract was
evaporated at reduced pressure and redissolved
in 0.7 ml of
deuterochloroform for
13C-NMR analysis.
Analytical methods.
Capillary GC-FID analysis of the PACs
remaining in the cultures and GC-mass-spectrometry analysis of
metabolites were conducted as described previously (5, 6).
Broad-band-decoupled
13C-NMR spectroscopy experiments were
carried out with a GE-QE 300Plus or a Nicolet NT-300-WB NMR
spectrometer
at a resonance frequency of 75.61 MHz. The
13C-NMR spectra in experiments with labeled acenaphthene
were acquired
in 2,400 scans, using a 31° pulse width and a 1-s
recycle delay
time.
1H- and
13C-NMR spectra for
the identification of synthetic materials and
comparison with those of
authentic compounds were recorded on
the same spectrometers.
Deuterochloroform was used as the solvent.
Tetramethylsilane (0.00 ppm)
and the central signal of the CDCl
3 (77.0) ppm were used as
reference signals.
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RESULTS AND DISCUSSION |
Pure-culture incubations with naphthalene.
Experiments on
degradation of [1-13C]acenaphthene in the presence of
naphthalene by the individual naphthalene- and
phenanthrene-degrading strains BR or BC were used to establish the
experimental and analytical parameters needed to carry out
13C-NMR spectroscopy studies under more diverse chemical
and microbiological scenarios. Although neither strain BR nor BC could
grow on acenaphthene as the sole carbon and energy source, acenaphthene
was oxidized by these strains when naphthalene was added as a
cosubstrate. In the presence of naphthalene,
[1-13C]acenaphthene was oxidized by strains BR and BC,
with naphthalene no longer detectable after 24 h. Only partial (30 to 40%) depletion of acenaphthene occurred during the first 5 days of
incubation. No further degradation of acenaphthene was observed in
longer incubations.
13C-NMR spectroscopy analyses showed that oxidation of
[1-
13C]acenaphthene under these conditions was
accompanied by accumulation
of several extractable
13C-labeled biotransformation products, as evidenced by the
appearance
of new resonances (Fig.
1;
Table
1). Based on chemical shift
data
and comparison with
13C-NMR data for authentic unlabeled
compounds, these resonances
were assigned to the acenaphthene oxidation
products marked B
through G in Fig.
2.
Resonance B was tentatively assigned to
[2-
13C]acenaphthen-1-ol or
[2-
13C]acenaphthen-1-one, both of which had
C-2 resonance frequencies
at approximately 42.1 ppm.
Resonances E (
C-1 = 74.4 ppm) and
D (
C-1 = 73.2 ppm) were assigned to [1-
13C]acenaphthen-1-ol
and [1-
13C]acenaphthen-1,2-diol, respectively. The
stereochemistry of [1-
13C]acenaphthen-1,2-diol could not
be unequivocally assigned since
the authentic
cis- and
trans-diols produce nearly coincidental
signals for each of
the
sec-alcohol carbon atoms. Resonance F
(169.4 ppm) was
indicative of a compound with a labeled carboxyl
group and was assigned
to the anhydride of [1-
13C]naphthalene-1,8-dicarboxylic
acid (formed from the free acid
under the acidic conditions of
extraction). Resonance G (
C-1 = 203.0 ppm) was due to
the
13C-labeled carbonyl carbon in
[1-
13C]acenaphthen-1-one. As indicated by the intense
resonance A,
[1-
13C]acenaphthene (
C-1 = 30.3 ppm) was still present in the cultures.
With respect to the
chemical nature of the compound responsible
for resonance C (
= 51.1 ppm), a definitive assignment cannot
be made at present. None of the
known acenaphthene metabolites
identified as products of initial
reactions catalyzed by bacteria
or fungi (
12,
14,
16,
19)
can account for a
13C chemical shift value of a carbon atom
derived from the labeled
benzylic atom of acenaphthene. One possible
explanation for resonance
C is that it is from an unidentified compound
formed by bacterial
oxidation, and possibly cleavage, of one of the
aromatic rings
of [2-
13C]acenaphthen-1-one. A chemical
shift of 51.1 ppm could be due
to a
13C-labeled
sp3 carbon located between a carbonyl group and
an aromatic ring
or between two carbonyl groups of a
-diketone.
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FIG. 1.
Representative 13C-NMR spectra of
extractable compounds obtained in biodegradation experiments with
Pseudomonas sp. strain BR after 2 days of incubation of
[1-13C]acenaphthene (20% 1-13C, 10 mg/25 ml)
and naphthalene (10 mg/25 ml) (A) and after 7 days of incubation of
creosote PACs (25 mg) spiked with 1 mg of
[1-13C]acenaphthene (B). For assignment of resonances A
to G, see Fig. 2. T, tetramethylsilane; S, solvent (CDCl3);
N, signals due to the natural abundance of aromatic
[13C]carbon atoms in acenaphthene and creosote PACs.
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TABLE 1.
Broad-band-decoupled 13C-NMR analysis of
[1-13C]acenaphthene and extractable labeled metabolites
formed by the aromatic hydrocarbon-degrading microorganisms in time
course biodegradation experiments
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FIG. 2.
Assignment of 13C-NMR signals to detected
metabolites of the pathway for acenaphthene degradation by CREOMIX
culture and individual strains BR and BC.  , 13C-labeled
carbons.
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Recently, a key enzyme of bacterial naphthalene catabolism, naphthalene
1,2-dioxygenase, has been shown to catalyze monooxygenation
of benzylic
methylenic groups of acenaphthene. A series of acenaphthene
oxygenation
products are formed by strains that express genes
encoding this enzyme
(
16). These products include acenaphthen-1-ol,
acenaphthenone, both
cis- and
trans-acenaphthene-1,2-diols, and
naphthalene-1,8-dicarboxylic acid (recovered as its anhydride).
The
same range of products formed from [
13C]acenaphthene by
naphthalene-grown cells of strains BR and BC
(Fig.
2) indicates that
these labeled metabolites are likely to
be formed in reactions
initiated via monooxygenation of acenaphthene
by the naphthalene
dioxygenases of these strains.
When acenaphthene-grown cells of strain A2279 were incubated with
[1-
13C]acenaphthene in the presence of naphthalene, GC
analysis revealed
that both hydrocarbons were completely depleted
within 24 h. None
of the metabolites formed by strains BC and BR
was detected by
13C-NMR spectroscopy or GC-mass
spectrometry (data not shown). Evidently,
enzymes specific for
acenaphthene catabolism convert this substrate
to central cell
metabolites without product accumulation.
Pure-culture incubations with creosote PACs.
Individual
bacterial strains BC and BR, in the presence of creosote PACs, produced
essentially the same set of oxidation products from
[1-13C]acenaphthene as those observed in experiments with
[1-13C]acenaphthene in the presence of naphthalene. The
relative amounts of products were different, however (Fig. 1B; Table
1). Despite the complexity of the substrate mixture introduced by using
creosote PACs, the biodegradation reactions performed by these strains led to the accumulation of the same "dead-end" oxidation products. Formation of the same oxidation end products in this series and in the
experiments described above implies that limited oxidation of
acenaphthene by naphthalene dioxygenase, or a closely related oxygenase
system, may also occur when acenaphthene is biodegraded in the presence
of PAC mixtures such as are encountered in creosote. Substantial
amounts of acenaphthene (40 to 50% of the initial levels) remained
even after 2 weeks of incubation, indicating the relatively inefficient
oxidation of this compound under these conditions.
The action of the individual acenaphthene-catabolizing strain A2279 on
[1-
13C]acenaphthene in the presence of creosote PACs was
also examined.
Although the ability of this organism to degrade PACs
other than
acenaphthene and naphthalenes is limited (Table
2), no accumulation
of
13C-labeled acenaphthene biodegradation products was
detected by
NMR spectroscopy analysis during growth on creosote PACs
supplemented
with [1-
13C]acenaphthene (Table
1). As
discussed previously (
3), the
limited microbial diversity of
cultures used in degradation of
hydrocarbon mixtures may be a factor
leading to elevated levels
of biodegradation end products. For example,
biodegradation of
an artificially weathered oil by defined cocultures
of microbial
isolates resulted in the accumulation of larger amounts of
organic
acidic and neutral products than the amounts accumulated by
more
diverse undefined mixed cultures established by an enrichment
on
oil (
3).
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TABLE 2.
Concentration of selected PACs during biodegradation
experiments with creosote PACs spiked
with [1-13C]acenaphthene
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Undefined mixed-culture incubations with creosote PACs.
A
series of experiments with the undefined mixed bacterial culture
CREOMIX was undertaken to establish whether the formation of
acenaphthene oxidation products is due to the limited catabolic versatility of the individual strains used or is observable when PACs
are degraded by more complex cultures. Biodegradation of creosote PACs,
spiked with [1-13C]acenaphthene, by the CREOMIX
culture resulted in degradation of all aromatic compounds with three or
fewer rings (Table 2). Significant volatility of naphthalene,
monomethylnaphthalenes, and biphenyl had also contributed to the
observed depletion of these three compounds, as evidenced by the data
shown for the 14-day control incubation. However, effective
biodegradation of these compounds by the CREOMIX culture was evident,
since their levels were reduced severalfold within first 3 days and
completely depleted by day 7 of incubation. Only limited degradation of
two PACs with four rings, fluoranthene and pyrene, was observed.
13C-NMR spectroscopy analysis of the total extractable
compounds showed the presence of resonances corresponding to several of the acenaphthene oxidation products previously detected in experiments with the individual strains BR and BC (Table 1; Fig.
3A to C). The 13C-NMR spectra
showed the presence of [1-13C]- and
[2-13C]acenaphthen-1-ones, naphthalene 1,8-dicarboxylic
acid anhydride, and the same unidentified product described above with
a labeled methylenic group (resonance C at 51.1 ppm). In addition to
these products, traces of carboxyl-labeled carboxylic acids (minor
resonances with around 170 ppm) and of a product with a labeled
methylenic group ( ~ 41.7 ppm) were observed after 14 days of
incubation (Fig. 3C).
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FIG. 3.
Representative 13C-NMR spectra (expanded
baseline region) of extractable compounds obtained in biodegradation
experiments with creosote PACs (25 mg) spiked with 1 mg of
[1-13C]acenaphthene. (A) Day zero; (B) CREOMIX culture, 3 days of incubation; (C) CREOMIX culture, 14 days of incubation; (D)
CREOMIX culture plus Pseudomonas sp. strain A2279, 3 days of
incubation; (E) CREOMIX culture plus Pseudomonas sp. strain
A2279, 14 days of incubation. For assignment of the signals, see Fig. 2
and the legend to Fig. 1.
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The range of accumulated
13C-labeled products in
experiments with the CREOMIX culture indicates that biodegradation of
acenaphthene
in a creosote PAC mixture also occurs via oxidation of
benzylic
methylenic groups but is not limited to these reactions.
Additional
reactions responsible for the formation of more extensively
oxidized
metabolites, such as aromatic ring oxidation products, that
were
not observed in experiments with individual strains may have
occurred
due to the action of other strains present in the CREOMIX
culture.
However, even in these biodegradation experiments with mixed
cultures
presumed to provide an extended biochemical diversity,
acenaphthene
biodegradation was not complete after 14 days of
incubation. Because
acenaphthene-utilizing strains could not be
isolated from the
CREOMIX culture by subculturing with acenaphthene as
the sole
carbon source (see Materials and Methods), this compound is
evidently
degraded to only a limited extent via biotransformations
initiated
by naphthalene dioxygenase (or phenanthrene dioxygenase)
rather
than by a more complete growth-based process.
Coinoculation of the acenaphthene-grown strain A2279 with the CREOMIX
culture resulted in accelerated biodegradation of creosote
PACs
supplemented with [1-
13C]acenaphthene. Nearly complete
depletion of all aromatic compounds
with three or fewer rings was
achieved within the first 7 days
of incubation (Table
2). In contrast
to the experiments where
the CREOMIX culture was acting alone (Fig.
3B
and C), resonance
A, due to the
13C-labeled methylenic
group of acenaphthene, was no longer detectable
by
13C-NMR
spectroscopy after 14 days of PAC biodegradation by a coculture
of
CREOMIX and strain A2279 (Table
1; Fig.
3E). At all sampling
times, the
13C-NMR spectra of the compounds extracted from the
coinoculated
cultures revealed no distinct resonances due to the
labeled acenaphthene
metabolites described above and therefore
demonstrated that these
products do not accumulate under this
biodegradation scenario
(Table
1; Fig.
3D and E).
The results obtained demonstrate that mixed bacterial cultures,
established in the laboratory by enrichments on creosote PACs,
have a
restricted biochemical diversity with respect to furnishing
an
efficient pathway for acenaphthene degradation and catabolism.
It would
appear that the enrichment technique with creosote PACs
used here does
not provide favorable conditions for establishing
bacterial populations
which contain bacterial strains capable
of mineralizing acenaphthene.
Acenaphthene-utilizing strains,
such as A2279 and the
Alcaligenes strains reported previously
(
19),
could readily be isolated from soils contaminated with
coal-derived
products by enrichments with acenaphthene. Although
biochemical
pathways for acenaphthene catabolism have not been
established in
detail, they are clearly distinct from those of
naphthalene and
phenanthrene utilization and require enzyme systems
for the conversion
of key intermediates such as naphthalene-1,8-dicarboxylic
acid. It is
possible that the presence of large quantities of
naphthalene and
phenanthrene in creosote PACs provides more favorable
conditions for
the selection and dominance of bacterial strains
that effectively
catabolize these constituents, rather than the
less abundant
acenaphthene. This, however, does not completely
explain why
acenaphthene-utilizing strains are not evident in
the CREOMIX culture,
since acenaphthene is not completely removed
even after long incubation
times. Regardless of the explanation,
this study demonstrates that the
variety of substrates present
in complex mixtures of fossil
fuel-derived materials demands a
biochemically diverse and versatile
microbial flora for their
extensive biodegradation.
Another set of conclusions can also be drawn from comparison of the
13C-NMR (Table
1) data with those obtained by GC-FID
analysis (Table
2), a method more routinely used to quantify the
depletion of
key PAC analytes. Although no special efforts were made at
this
point to acquire quantitative NMR data on the compounds of
interest,
the decrease in the area of the resonance due to the
13C-labeled carbon of [1-
13C]acenaphthene
(resonance A) is correlated with acenaphthene depletion
as measured by
GC-FID, with an error not exceeding 10%. At the
same time, however,
changes in concentrations of accumulated metabolites
together with
structural information could also be shown by NMR
spectroscopy, while a
typical GC-FID protocol is not satisfactory
for this purpose. It is
also important to note that, based on
the semiquantitative estimates of
this study, as little as 2 to
3% of the introduced
13C
label was readily detected in intermediates or biodegradation
end
products by
13C-NMR spectroscopy. This observation is
significant because it
points out that very little
13C-labeled material is needed to obtain useful structural
information.
The results of this study demonstrate the utility of
13C
labeling in combination with standard NMR spectroscopy experiments
for
studies of the biodegradation of complex mixtures of compounds,
such as
coal-derived wastes. The approach allows for direct analysis
of
stable-isotope-labeled products and, in this case, an assessment
of the
course of the biodegradation process. NMR spectroscopy
is a recognized
structural analytical tool, and when it is used
in combination with
site-specific
13C labeling, the enhanced sensitivity
provided by the labeled carbon(s)
serves as a means to track the fate
of a chosen substrate and
the chemical changes which occur at, or in
the vicinity of, the
label. Further successful application of the
method to the biodegradation
of a diverse spectrum of fossil-derived
materials depends on the
availability of relevant
13C
tracer compounds. When chosen in accordance with their abundance
in the
mixtures of interest and labeled in positions which are
relevant to
available information on their biodegradation pathways,
such labeled
compounds can serve as a diversified collection of
specific tracers for
a variety of fossil fuel materials. The use
of this collection, in
conjunction with NMR spectroscopy and other
sensitive methods such as
isotope ratio MS, can provide an invaluable
tool for the identification
of the biochemical limitations of
biodegradation processes, for the
evaluation of bioremediation
performance, and for the analysis of
pollutant and metabolite
fate in complex organic matrixes such as soils
and sediments.
| |
ACKNOWLEDGMENTS |
This work was supported in part by a grant from the Office of
Naval Research, U.S. Navy (N00014-95-1-0209), and by Cooperative Agreement CR-823946-01-0 with U.S. EPA. The initial stages of the work
were also supported by Cooperative Agreement CR-817770 with U.S. EPA.
We gratefully acknowledge Charylene Gatlin (University of West Florida)
for assistance with microbiological experiments and Sol Resnick
(formerly Technical Resources, Inc., Gulf Breeze, Fla.) for isolation
of the microorganisms.
| |
FOOTNOTES |
*
Corresponding author. Present address: Maxygen, Inc.,
3140 Central Expressway, Santa Clara, CA 95051. Phone: (408) 522-6083. Fax: (408) 732-4558. E-mail: Sergey_Selifonov{at}maxygen.com.
Contribution no. 1021 from the Gulf Ecology Division, NHEERL,
U.S. Environmental Protection Agency, Gulf Breeze, Fla.
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0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
