Previous Article | Next Article 
Applied and Environmental Microbiology, October 2001, p. 4842-4849, Vol. 67, No. 10
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.10.4842-4849.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Stable Hydrogen and Carbon Isotope Fractionation
during Microbial Toluene Degradation: Mechanistic and Environmental
Aspects
Barbara
Morasch,1
Hans H.
Richnow,2
Bernhard
Schink,1 and
Rainer U.
Meckenstock1,*
Lehrstuhl für Mikrobielle
Ökologie, Universität Konstanz, D-78457
Konstanz,1 and Sektion
Sanierungsforschung, Umweltforschungszentrum Leipzig-Halle GmbH,
D-04318 Leipzig,2 Germany
Received 29 January 2001/Accepted 16 July 2001
 |
ABSTRACT |
Primary features of hydrogen and carbon isotope fractionation
during toluene degradation were studied to evaluate if analysis of
isotope signatures can be used as a tool to monitor biodegradation in
contaminated aquifers. D/H hydrogen isotope fractionation during microbial degradation of toluene was measured by gas chromatography. Per-deuterated toluene-d8 and
nonlabeled toluene were supplied in equal amounts as growth substrates,
and kinetic isotope fractionation was calculated from the shift of the
molar ratios of toluene-d8 and
nondeuterated toluene. The D/H isotope fractionation varied slightly
for sulfate-reducing strain TRM1 (slope of curve [b] = 
1.219), Desulfobacterium cetonicum
(b = 1.196), Thauera aromatica
(b = 0.816), and Geobacter
metallireducens (b = 1.004) and was
greater for the aerobic bacterium Pseudomonas putida
mt-2 (b = 2.667). The D/H isotope fractionation
was 3 orders of magnitude greater than the
13C/12C carbon isotope fractionation reported
previously. Hydrogen isotope fractionation with nonlabeled toluene was
1.7 and 6 times less than isotope fractionation with per-deuterated
toluene-d8 and nonlabeled toluene for
sulfate-reducing strain TRM1 (b = 0.728) and
D. cetonicum (b = 0.198),
respectively. Carbon and hydrogen isotope fractionation during toluene
degradation by D. cetonicum remained constant over a
growth temperature range of 15 to 37°C but varied slightly during
degradation by P. putida mt-2, which showed maximum
hydrogen isotope fractionation at 20°C (b = 4.086) and minimum fractionation at 35°C (b = 2.138). D/H isotope fractionation was observed only if the deuterium
label was located at the methyl group of the toluene molecule which is
the site of the initial enzymatic attack on the substrate by the
bacterial strains investigated in this study. Use of ring-labeled
toluene-d5 in combination with
nondeuterated toluene did not lead to significant D/H isotope fractionation. The activity of the first enzyme in the anaerobic toluene degradation pathway, benzylsuccinate synthase, was measured in
cell extracts of D. cetonicum with an initial activity
of 3.63 mU (mg of protein)
1. The D/H isotope
fractionation (b = 1.580) was 30% greater than that in growth experiments with D. cetonicum. Mass
spectroscopic analysis of the product benzylsuccinate showed that H
atoms abstracted from the toluene molecules by the enzyme were retained
in the same molecules after the product was released. Our findings
revealed that the use of deuterium-labeled toluene was appropriate for studying basic features of D/H isotope fractionation. Similar D/H
fractionation factors for toluene degradation by anaerobic bacteria,
the lack of significant temperature dependence, and the strong
fractionation suggest that analysis of D/H fractionation can be used as
a sensitive tool to assess degradation activities. Identification of
the first enzyme reaction in the pathway as the major fractionating
step provides a basis for linking observed isotope fractionation to
biochemical reactions.
| |
INTRODUCTION |
Biological isotope
fractionation leads to unequal distribution of heavier and lighter
isotopes between the educts and products of a reaction. In most cases,
the lighter isotopes are preferentially used and the heavier isotopes
are enriched in the residual substrate fraction. This is a well-known
phenomenon for CO2 fixation during photosynthesis
(25) and methanogenesis (11, 17) and for methane oxidation (5, 8, 36). In recent years, isotope fractionation has been described for bacterial degradation of environmental contaminants, such as chlorinated hydrocarbons (6, 10, 16) and aromatic compounds (1, 18, 21, 22, 32, 38). In addition,
13C/12C isotope
fractionation of chlorinated and aromatic hydrocarbons was demonstrated
in contaminated aquifers (9, 16, 29), and this finding was
taken as an indication of microbial degradation. Assessment of isotope
fractionation was therefore discussed as a method for monitoring
biological degradation directly in the aquifer. However, in natural
environments factors like redox conditions and temperature can vary and
may influence isotope fractionation. To assess the contributions of
bacterial degradation activities in situ to natural
attenuation, further understanding of the possible effects
of physical and chemical parameters on isotope fractionation has to be attained.
In addition to carbon isotope fractionation of organic contaminants,
some studies have reported on chlorine (34) or
deuterium/hydrogen isotope fractionation (8, 35). Kinetic
D/H isotope fractionation has been shown to be 2 orders of magnitude
greater than carbon isotope fractionation (8). However,
single-compound isotope analysis by gas
chromatography-combustion-isotope ratio monitoring mass spectrometry
(GC-C-IRMS), which is the standard method used for carbon isotope
analysis (12, 23), became available for hydrogen
fractionation only recently (14), and hydrogen isotope analysis is still more expensive and less precise than carbon isotope analysis.
Here, we describe a method for investigating D/H isotope fractionation
during bacterial toluene degradation by gas chromatography (GC). The
depletion of lighter toluene species and the enrichment of labeled
toluene in the residual fraction were determined to assess hydrogen
isotope fractionation. The effects of different electron acceptors or
temperatures on isotope fractionation were checked, and the major
fractionating step in anaerobic bacterial toluene degradation was
identified. Furthermore, the proposed reaction mechanism of
benzylsuccinate synthase was confirmed by D/H isotope analysis with GC
and mass spectroscopy.
| |
MATERIALS AND METHODS |
Strains and cultivation.
Sulfate-reducing strain TRM1 was
isolated in our laboratory (20), and
Desulfobacterium cetonicum DSM 7267, Thauera
aromatica K172 (= DSM 6984), and Geobacter
metallireducens GS-15 (= DSM 7210) were purchased from the
Deutsche Sammlung von Mikroorganismen und Zellkulturen (Braunschweig,
Germany). Pseudomonas putida mt-2 was a kind gift from
J. R. van der Meer, Dübendorf, Switzerland.
Anaerobic strains were cultivated in carbonate-buffered freshwater
mineral medium, pH 7.2 to 7.4 (37). This medium was
prepared in the absence of oxygen under an
N2-CO2 atmosphere (80:20)
and was reduced with Na2S (1 mM). Either
Na2SO4 (10 mM) or
NaNO3 (10 mM) was added as an electron acceptor.
FeCl2 (3 mM) was added to the medium for strain
TRM1 in order to scavenge the sulfide produced. The same freshwater
medium was used to cultivate Geobacter metallireducens, but
FeCl2 (1 mM) was added as a reducing agent instead of sulfide and Fe(III) citrate (50 mM) was used as an electron
acceptor. Aerobic bacteria were cultivated in mineral medium M9
(30). Serum bottles (120 and 500 ml) were half filled with
mineral medium and tightly sealed with Viton rubber stoppers (Maag
Technik, Dübendorf, Switzerland). Toluene was injected into the
culture bottles with syringes through the stoppers and was allowed to
equilibrate overnight before inoculation. All substrates, including
nonlabeled toluene, per-deuterated
toluene-d8 (Fluka, Buchs, Switzerland),
ring-deuterated toluene-d5, and
methyl-deuterated toluene-d3 (Campro
Scientific, Berlin, Germany), were analytical grade. Growth experiments
were performed in triplicate at 30°C unless indicated otherwise.
Aerobic cultures were shaken at 100 strokes
min1.
Analysis and sampling.
Growth of sulfate-reducing bacteria
and G. metallireducens was monitored by measuring the
formation of sulfide or Fe(II) (7, 33). Growth of all
other strains was determined by measuring the increase in optical
density at 578 nm. Toluene concentrations were analyzed by
high-performance liquid chromatography (System Gold; Beckman,
Fullerton, Calif.) with a C18 reversed-phase
column (GROM-SIL; ODS-5 ST; length, 250 mm; particle size, 5 µm; Grom, Herrenberg, Germany) and UV detection (206 nm), using
acetonitrile-100 mM ammonium phosphate buffer (pH 3.5) (70:30,
vol/vol) as the eluent. Culture samples (250 µl) were diluted 1:5
with ethanol (99.9%, p. a.) and centrifuged (14,000 × g, 5 min) to remove precipitates.
Isotope analysis.
D/H isotope fractionation in experiments
with mixtures of deuterium-labeled toluene and nonlabeled toluene could
be assessed by separation of the different toluene species in a GC
capillary column. GC-C-IRMS is sensitive enough to detect changes in
the deuterium ratio in the per mille range and was used to analyze D/H
isotope fractionation in experiments with nonlabeled toluene. 13C/12C isotope
fractionation was also measured by GC-C-IRMS.
Liquid samples (2 to 7 ml) for GC analysis were taken with a syringe
through the stoppers and were extracted with 0.3 ml of
pentane
(Suprasolve; Merck, Darmstadt, Germany). Aliquots (4 µl)
of the
pentane phase were analyzed in three replicates with a
GC equipped with
a flame ionization detector (Carlo Erba Instruments,
Milan, Italy). The
GC was equipped with a fused silica capillary
column (optima
-3; length, 60 m; inside diameter, 0.32 mm; film
thickness, 0.35 µm; Macherey-Nagel, Düren, Germany). Baseline
separation of the different toluene species was achieved at 60°C
and
80 kPa with a N
2 flow rate of 1.5 ml
min
1. Samples were injected into the heated
injector (200°C) with
a split of 1:15.
13C/
12C isotope ratios were
determined by GC-C-IRMS. The system consisted of a GC (HP-5890;
Hewlett-Packard Co., San Diego, Calif.)
which was connected via a
combustion unit (GC-combustion interface;
Finnigan, Bremen, Germany) to
an isotope mass spectrometer (Finnigan
MAT 252; Finnigan). The GC was
equipped with a fused silica capillary
column (BPX-5; length, 50 m; inside diameter, 0.32 mm; film thickness,
0.5 µm; SGE, Darmstadt,
Germany). The temperature program consisted
of 2 min at 40°C , followed by a linear increase to 180°C at a
rate of 6°C
min
1. Samples were injected splitless at
250°C.
D/H isotope ratios were determined with an Isochrome GC-C-IRMS system
(Micromass, Manchester, United Kingdom) consisting of
a GC unit which
was connected via a chromium furnace to a mass
spectrometer (Isoprime,
Manchester, United Kingdom). The temperature
of the furnace was
adjusted to 1,800°C. Samples were injected
splitless at 20°C in a
KAS4 cooled on column injector (Gerstel,
Germany), and
subsequently the injector was heated to 300°C at
a rate of 12°C
s
1; the temperature was kept at 300°C for 5 min. The GC conditions
were the same as those described above
for determination of carbon
isotope
ratios.
Calculations.
Calculations were based on the Rayleigh
equation for closed systems, which was developed to describe
distillation processes (28) and was adapted for isotope
fractionation (equation 1) (15). D/H isotope fractionation
with labeled toluene was determined by calculating the GC peak areas of
pentane-extracted toluene species. The hydrogen isotope ratio
(Rt) was the quotient of the concentrations of toluene
([toluene-d8]/[toluene]) at time
t. If the logarithms of the isotope ratios
(Rt/R0)
were plotted over the respective logarithms of the concentrations
(ct/c0),
according to equation 2 (15, 22, 29), the kinetic isotope
fractionation factor (
) could be determined from the slopes of the
curves (b), with b = 1/ 1 (equation 3). ct and
c0 were the total toluene concentrations at time t and time zero, respectively. When
only nondeuterated toluene was supplied in isotope fractionation
experiments, isotope ratios (Rt) were
determined from the common values with equations 4 and 5
(22). The international PDB and SMOW standards were
used to calculate values from GC-C-IRMS analysis data. 0 was the initial isotope signature of the
substrate.
|
(1)
|
|
(2)
|
|
(3)
|
|
(4)
|
|
(5)
|
The fractionation factor (
) is a nonlinear function of
b given in
= 1/(
b + 1) and not defined
if
b =
1. For carbon isotope
fractionation
during toluene degradation,
b usually was between
0.001
and
0.01, a range where
is almost linearly dependent
on
b. The slopes of D/H isotope fractionation were within a
range
of
5 to 0. For
b approaching
1,
diverged
against ±
and became
an abstract term for quantitative
descriptions. It is therefore
more illustrative to describe D/H
fractionation by the slope,
which is used throughout this paper. The
slope decreases with
increasing isotope fractionation. The results of
13C/
12C isotope
fractionation experiments are given in the common
notation.
Benzylsuccinate synthase assay.
A culture of D. cetonicum (0.8 liter; optical density at 578 nm, 0.3) was
harvested in the late exponential growth phase. The cells were washed
in anoxic potassium phosphate buffer (50 mM; pH 7.5) which had been
supplemented with NaCl (175 mM) and MgCl2 (25 mM), reduced with H2 (105
Pa)-palladium-charcoal (1 mg liter1) (Merck,
Darmstadt, Germany) and titanium(III) citrate (1 mM), and filtered.
Cells were suspended in 3 ml of buffer and broken with a French
pressure cell (9 MPa) in the presence of 2 mM fumarate. Cell debris was
removed by centrifugation (14,000 × g, 5 min). The
cell extract was diluted to a volume of 6 ml with buffer. One
milliliter of a solution containing 0.5 mM
toluene-d8 and 0.5 mM toluene (each in
reduced potassium phosphate buffer) was added to the diluted extract,
and the preparation was mixed.
The benzylsuccinate synthase test was performed at 30°C in 8-ml glass
vials sealed with Viton rubber stoppers. At each sampling
time, 500 µl of buffer was injected into the reaction vial and
mixed.
Subsequently, a 500-µl sample was removed with a syringe
through the
stopper, and aliquots (200 µl) of the sample were
transferred into
two 1.7-ml glass vials. The reaction was stopped
with ice-cold 1 M NaOH
(20 µl), and the mixture was overlaid with
100 µl of pentane
(Suprasolve) containing 2.5 mM ethylbenzene
(Fluka, Buchs, Switzerland)
as an internal standard. A control
assay was run without cell extract.
Toluene-
d8/toluene ratios
and overall
toluene concentrations were determined by GC as described
above. The
aqueous phase remaining from the extraction was diluted
fivefold with
ethanol, and benzylsuccinate was analyzed by high-performance
liquid
chromatography as described above with acetonitrile-ammonium
phosphate
buffer (30:70) as the eluent. Protein concentrations
were determined
with a protein assay kit (Bio-Rad, Munich, Germany).
The identity of
benzylsuccinate in the extracts was confirmed
by coelution with a
benzylsuccinate reference (Sigma, Deisenhofen,
Germany).
Mass spectroscopic analysis.
Benzylsuccinate was converted
to its methyl ester with trimethylchlorosilane (Supelco, Bellefonte,
Pa.) in methanol (8:1, vol/vol) at 60°C for 1 h. The reaction
mixture was extracted with dichloromethane for a mass spectrometric
analysis performed with a quadrupole system (MSD; Hewlett-Packard Co.).
The GC was equipped with a fused silica capillary column (DB-1; length,
30 m; inside diameter, 0.32 mm; film thickness, 0.25 µm; J&W
Scientific, Folsom, Calif.). The injection mode was splitless.
The temperature program was as follows: 2 min at 60°C, followed by an
increase to 250°C at a rate of 4°C min1.
| |
RESULTS |
Separation of isotopic toluene species.
A method was developed
to determine D/H isotope fractionation upon toluene degradation by GC
analysis. Batch cultures were grown with mixtures of deuterated
toluene-d8 and nonlabeled toluene (50:50,
vol/vol). The nondegraded residual toluene fraction in the cultures was
extracted with pentane and analyzed by GC. The different toluene
species were separated by GC; the elution time for
toluene-d8 was 13.8 min, and this compound
was followed by methyl-labeled toluene-d3
(13.95 min), ring-labeled toluene-d5 (14.0 min), and nonlabeled toluene (14.2 min). During growth with a mixture
of per-deuterated toluene-d8 and
nonlabeled toluene, the different bacterial strains degraded nonlabeled
toluene faster, and consequently the hydrogen isotope ratios
(Rt) of
[toluene-d8] to [toluene] increased
substantially, as illustrated by a degradation experiment performed
with D. cetonicum (Fig. 1).
View larger version (15K):
[in this window]
[in a new window]
|
FIG. 1.
Substrate conversion during growth of D.
cetonicum with a mixture of 50%
toluene-d8 and 50% nonlabeled toluene
as the sole carbon and energy source. Toluene concentrations ( ) and
related toluene-d8/toluene ratios in
the residual nondegraded substrate fraction ( ) were monitored over
time. Rt is the isotope ratio
calculated as follows:
[toluene-d8]/[toluene].
|
|
D/H isotope fractionation factors for various bacterial strains
cultivated with toluene.
The D/H isotope fractionation factors
obtained in growth experiments with different bacterial strains were
all of the same order of magnitude. The greatest fractionation for
toluene degradation was observed in growth experiments with the aerobic
bacterium P. putida mt-2, with fractionation of
b = 2.667 ± 0.163. The D/H isotope
fractionation by anaerobic bacteria was slightly less, with
b = 1.219 ± 0.254 for sulfate-reducing strain
TRM1, b = 1.196 ± 0.075 for D. cetonicum, b = 1.004 ± 0.077 for G. metallireducens, and b = 0.816 ± 0.133 for
T. aromatica (Fig. 2). All of
the anaerobic strains showed similar degrees of isotope fractionation,
although they used different electron acceptors.
View larger version (15K):
[in this window]
[in a new window]
|
FIG. 2.
D/H isotope fractionation during degradation of a
mixture of toluene-d8 and nonlabeled
toluene in batch cultures. Representative curves from three replicates
are shown for P. putida mt-2 ( ), strain TRM1 ( ),
D. cetonicum (), G. metallireducens
( ), and T. aromatica ( ). For D.
cetonicum () and strain TRM1 ( ) D/H isotope fractionation
was determined in batch cultures grown with nonlabeled toluene, and the
natural abundance of deuterium was measured by GC-C-IRMS.
|
|
D/H isotope fractionation of toluene with the natural deuterium
abundance.
D. cetonicum and strain TRM1 were grown with
nonlabeled toluene, and the D/H isotope compositions were determined by
GC-C-IRMS. D/H isotope fractionation of b = 0.728 was
obtained for strain TRM1 with nonlabeled toluene; this fractionation
was 1.7 times less than the fractionation observed when
toluene-d8 and nonlabeled toluene were
supplied. In experiments with D. cetonicum grown with
nonlabeled toluene, the D/H isotope fractionation was b = 0.198, or six times less than the value obtained in growth
experiments with per-deuterated toluene-d8
and nonlabeled toluene (50:50) (b = 1.196) (Fig. 2),
indicating that isotope fractionation with deuterated toluene was not
identical to fractionation with nonlabeled toluene but was of the same
order of magnitude.
Temperature dependence of isotope fractionation.
To assess the
effects of temperature on isotope fractionation, bacterial D/H isotope
fractionation upon toluene degradation was investigated in growth
experiments performed with the aerobic bacterium P. putida
mt-2 and per-deuterated toluene-d8 and
nonlabeled toluene in equal amounts at five different temperatures
between 15 and 35°C. The highest isotope fractionation value was
obtained at 20°C, with b = 4.086 ± 0.127; the
value decreased to b = 2.138 ± 0.667 at 35°C
(Fig. 3A). With the anaerobic bacterium
D. cetonicum, the D/H isotope fractionation value differed
between b = 1.092 ± 0.239 at 18°C and
b = 1.260 ± 0.009 at 37°C, a difference which was not significant within the range of the standard deviation (Fig.
3A).
View larger version (15K):
[in this window]
[in a new window]
|
FIG. 3.
Influence of temperature on D/H isotope fractionation
(A) and 13C/12C isotope fractionation (B)
during toluene degradation by D. cetonicum (),
sulfate-reducing strain TRM1 (), and P. putida mt-2
(). (A) D/H isotope fractionation factors were determined by
determining the slope (b = 1/ 1) of the
double-logarithmic plot of equation 2. For D/H isotope fractionation,
each data point represents the average fractionation factor calculated
from three independent growth experiments; the error bars indicate the
standard deviations. (B) Carbon isotope fractionation factors were
calculated from the regression curves in a double-logarithmic plot of
equation 2. Each data point represents a
13C/12C isotope fractionation factor (C).
The error bars indicate the reliability of C calculated from the
slopes (b = 1/ 1) of the regression
curves.
|
|
Temperature effects on carbon isotope fractionation were analyzed in
growth experiments performed with
P. putida mt-2,
D. cetonicum, and strain TRM1 cultivated with nonlabeled toluene.
Similar to D/H isotope fractionation, there was a slight decrease
in
the
13C/
12C isotope
fractionation factors with decreasing temperatures for
P. putida mt-2 from
C = 1.0042 ± 0.0006 at 15°C to
C = 1.0025
± 0.0003 at 35°C. The
13C/
12C isotope
fractionation factors for the anaerobic organisms
D. cetonicum and TRM1, however, did not vary with temperature within
the observed standard deviations (Fig.
3B).
Identification of the fractionating step.
We examined whether
the observed strong D/H isotope fractionation during bacterial toluene
degradation could be attributed to the first enzyme reaction of the
toluene degradation pathway or to other parameters, like substrate
diffusion to the cells or substrate uptake. Therefore, different
toluene species with deuterium labels either at the aromatic ring
(toluene-d5) or at the methyl group
(toluene-d3) were used as growth
substrates in equal amounts with per-deuterated
toluene-d8 or nondeuterated toluene. The
three bacteria used initiate degradation with an attack on the methyl
group; this has been proven for P. putida mt-2
(31) and D. cetonicum (24) and is
assumed for strain TRM1. D. cetonicum (Fig.
4), sulfate-reducing strain TRM1, and P. putida mt-2 showed strong D/H isotope effects
(b = 1.251, b = 1.280, and
b = 4.218, respectively) (Table
1) if methyl-deuterated toluene-d3 was used in combination with
nonlabeled toluene. When ring-deuterated
toluene-d5 was used in combination with
nonlabeled toluene, the D/H isotope fractionation factors for D. cetonicum, strain TRM1, and P. putida mt-2 were
negligible within the analytical errors. In addition, no D/H isotope
fractionation within the standard deviations could be demonstrated for
the three strains if methyl-deuterated toluene-d3 was provided in equal amounts
with per-deuterated toluene-d8. When
ring-deuterated toluene-d5 was used in
combination with per-deuterated toluene-d8, the D/H isotope fractionation
factors were b = 0.679 for D. cetonicum,
b = 0.917 for strain TRM1, and b = 2.696 for P. putida mt-2 and thus slightly lower than the
values obtained in experiments with
toluene-d3 and nonlabeled toluene.
View larger version (13K):
[in this window]
[in a new window]
|
FIG. 4.
D/H isotope fractionation during degradation of various
combinations of selectively deuterated toluene species. Representative
curves from three replicates are shown for D. cetonicum
grown with equal amounts of toluene-d8
and toluene-d5 (), with
toluene-d3 and nonlabeled toluene
(), with toluene-d8 and nonlabeled
toluene (), with toluene-d5 and
nonlabeled toluene (), and with
toluene-d8 and
toluene-d3 (). The curves were
plotted by using equation 2.
|
|
View this table:
[in this window]
[in a new window]
|
TABLE 1.
D/H isotope fractionation during growth of D. cetonicum, sulfate-reducing strain TRM1, and P. putida mt-2 with mixtures (50:50) of two selectively deuterated
toluene species
|
|
Benzylsuccinate synthase assay with D.
cetonicum.
The activity and D/H isotope fractionation of
benzylsuccinate synthase were determined in discontinuous enzyme assays
at 30°C. About 80 µM toluene was consumed, and 70 µM
benzylsuccinate was produced (Fig. 5). At
the beginning of the experiment the enzyme activity in the cell
extracts was 3.63 mU (mg of protein)1, but the
activity decreased to zero within 180 min. The maximum toluene turnover
rate determined was 12% of the in vivo degradation activity (29.3 nmol
min1 mg of protein1).
The D/H isotope fractionation obtained in the benzylsuccinate synthase
assay was b = 1.580 with 50%
toluene-d8 and 50% nonlabeled toluene as
the substrates, or 30% greater than the fractionation by growing
D. cetonicum cells (Fig. 6).
Mass spectroscopic analysis revealed that
benzylsuccinate-d8 and nondeuterated
benzylsuccinate were produced. The maximum rate of nondeuterated
benzylsuccinate production was 4.4 nmol min1 mg
of protein 1 and was 11 times higher than the
rate of production of benzylsuccinate-d8 (0.4 nmol min1 mg of
protein1). The mass peaks m/z 243 (benzylsuccinate-d7) and m/z
237 (benzylsuccinate-d1) could not be
detected.
View larger version (20K):
[in this window]
[in a new window]
|
FIG. 5.
Discontinuous benzylsuccinate synthase activity assay
with D. cetonicum cell extract. The total concentrations
of toluene-d8 and nonlabeled toluene
() and of benzylsuccinate () shown are means based on two samples
per time point. The specific benzylsuccinate synthase activity ( )
was calculated by determining the toluene degradation rate between two
adjacent sampling points.
|
|
View larger version (12K):
[in this window]
[in a new window]
|
FIG. 6.
Toluene D/H isotope fractionation in the cell-free
benzylsuccinate synthase reaction () and in a growth experiment
() with D. cetonicum. Equal amounts of
toluene-d8 and nonlabeled toluene were
used in both experiments.
|
|
| |
DISCUSSION |
Mechanistic aspects of hydrogen and carbon isotope fractionation
were studied to assess whether isotope fractionation could serve as a
tool to estimate biological degradation in contaminated aquifers.
Therefore, isotope fractionation was investigated with several
bacterial strains using different electron acceptors during toluene
degradation. The influence of growth temperature was determined, and
the fractionating step in anaerobic toluene degradation was identified.
Hydrogen isotope fractionation was studied by a method in which the
strains were grown on mixtures of different deuterated toluene species
under defined laboratory conditions. Thus, basic questions of bacterial
isotope fractionation could be determined by GC analysis without using
the more expensive and time-consuming technique of single-compound
isotope analysis by GC-C-IRMS.
Many biochemical reactions are known to cause significant partitioning
of isotopes between substrates and products. Isotope effects not only
are due to sheer mass differences but also are due to the impact of
additional neutrons on the mechanism and velocity of a biochemical
reaction. Direct involvement of a bond which is substituted with a
heavy isotope is known as a primary isotope effect. The dissociation
energy needed to cleave a heavy-isotope-substituted bond is higher and
is reflected in a decrease in the reaction rate (26).
Weaker, secondary isotope effects occur when the heavy-atom isotope is
located close to the bond but is not directly involved in the reaction mechanism.
D/H isotope fractionation factors for various bacterial strains
cultivated with toluene.
In contrast to carbon isotope
fractionation, which was almost the same for all of the anaerobic
strains tested (22), the 100- to 1,000-times-greater
hydrogen isotope fractionation factors varied among the different
species within the same order of magnitude. As the mass of deuterium is
100% greater than the mass of a hydrogen atom, deuterium isotope
effects are more pronounced. Small differences in the toluene-degrading
enzymes are multiplied by these effects and are reflected in the
variations in the D/H isotope fractionation factors for the bacterial
strains studied. These variations in D/H isotope fractionation are
unfavorable for quantitative evaluation of biological degradation at
natural sites by analysis of hydrogen isotope ratios. Nevertheless, as
the hydrogen isotope fractionation was at least 100-fold greater than
the corresponding carbon isotope fractionation, D/H isotope ratios
might be promising indicators for detection of low bacterial activities
in natural environments.
D/H isotope fractionation of toluene for natural deuterium
abundance.
D/H isotope fractionation by the
sulfate-reducing organisms strain TRM1 and D. cetonicum
grown with toluene-d8 and nonlabeled
toluene was compared to fractionation when nonlabeled toluene was used
as the sole carbon source, in order to determine the reliability of
using labeled toluene species in D/H isotope fractionation studies. The
isotope fractionation values determined for growth with nonlabeled
toluene were 1.7 and 6 times lower than the values determined for
growth with toluene-d8 and nonlabeled
toluene for strain TRM1 and D. cetonicum, respectively. The reason for this might be different reactivity of a methyl group
labeled with three deuterium atoms compared to the reactivity of
nonlabeled toluene molecules which have only one deuterium atom at the
methyl group. In the case of per-deuterated
toluene-d8 the primary isotope effect
resulting from cleavage of a C---D bond is enhanced by secondary
isotope effects caused by two additional deuterium atoms in the methyl
group. For statistical reasons the natural occurrence of toluene with
two or three deuterium atoms at the methyl group is negligible.
Another factor contributing to the differences in the results of
isotope fractionation between nonlabeled toluene and
toluene-
d8-nonlabeled
toluene is the two
types of isotope analysis used. Determination
of isotope fractionation
by GC is based on separation of methyl-labeled
toluene and
non-methyl-labeled toluene. Therefore, every deuterium
atom of the
labeled molecule is directly or indirectly subject
to fractionation.
GC-C-IRMS analysis of toluene for natural deuterium
abundance detects
every deuterated toluene molecule without taking
into account the
localization of the deuterium atoms. Statistically,
0.8% of all
toluene molecules carry a deuterium atom. The probability
that the
deuterium label is located at the methyl group is 3/8,
which means that
five-eighths of the molecules detected are not
subject to
fractionation. Thus, the isotope fractionation factors
determined with
labeled compounds should be two to three times
greater than the factors
obtained with nonlabeled toluene because
of the different methods used,
which indeed was the
case.
The relative concentrations of deuterium-labeled molecules did not
influence isotope fractionation; this conclusion was reached
because
the natural abundance of deuterium in nonlabeled toluene
was 4 orders
of magnitude lower than the abundance in experiments
performed with
deuterated toluene. The results indicate that the
use of
deuterium-labeled toluene to study D/H isotope fractionation
is a valid
technique, although the fractionation obtained is not
identical to the
fractionation obtained with nonlabeled
toluene.
Temperature dependence of isotope fractionation.
In
geosciences, temperature is a well-known parameter that affects the
degree of isotope fractionation (15, 26). We examined whether temperature alters the extent of isotope fractionation during toluene degradation. No significant dependence on
temperature for hydrogen or carbon isotope fractionation was
observed for the anaerobic bacteria D. cetonicum and strain
TRM1. In contrast, hydrogen isotope fractionation by P. putida mt-2 increased with decreasing temperature and showed the
greatest fractionation at 20°C. No clear effect of temperature on
13C/12C carbon isotope
fractionation by P. putida mt-2 could be found. The reason
why the temperature effect on isotope fractionation was so small might
be the temperature range of mesophilic bacterial activity (10 to
40°C), a more limited temperature range than that of geochemical
processes. It has been shown that undefined cultures of
methane-oxidizing bacteria exhibit greater
13C/12C isotope
fractionation at 30°C (C = 1.025) than at 11.5°C (C = 1.013) (8), although thermodynamics suggests that
fractionation decreases with increasing temperature. However, since in
this study an undefined mixture of methanotrophic bacteria was used, the observed effect might have been due to different subpopulations with alternate enzyme systems enriched at the two different
temperatures. Our results showed that variations in temperature should
not significantly affect isotope fractionation in contaminated anoxic
aquifers. There was also no correlation between toluene degradation
rates and isotope fractionation because growth of our strains was
closely linked with the growth temperature; e.g., the generation time of the sulfate-reducing strain TRM1 decreased from 25.5 days at 12°C
to 5 days at 30°C (data not shown), but the
13C/12C isotope
fractionation factors were identical (C = 1.0017).
Identification of the fractionating step.
Isotope
fractionation may be caused by transport of the substrate to the cell,
by uptake into the cell, or by the first enzyme reaction or a
subsequent enzyme reaction in the degradation pathway. Growth
experiments with selectively deuterated toluene species as carbon
sources were performed to identify which of the processes mentioned
above is relevant for fractionation. P. putida mt-2, D. cetonicum, and strain TRM1 all initiate toluene
degradation with an enzymatic attack on the methyl group (3, 4,
24, 31, 39). The greatest hydrogen isotope fractionation effects were observed if the bacteria grew with a mixture of methyl-labeled toluene-d3 or per-deuterated
toluene-d8 and nonlabeled toluene. If the
deuterium label was located on the aromatic ring or if toluene-d3 was used in combination with
per-deuterated toluene-d8, isotope
fractionation was not detectable. These findings indicate that higher
molecular masses did not influence processes such as transport to the
cells and substrate uptake and that the D/H isotope fractionation
determined was not due to the differences in the overall molecular
masses. Significant fractionation occurred only when the methyl group
was labeled, which shows that the initial enzymatic attack at the
methyl group was the major step which led to hydrogen isotope fractionation.
Benzylsuccinate synthase assay with D.
cetonicum
Benzylsuccinate synthase in a cell extract of
D. cetonicum exhibited 30% greater D/H isotope
fractionation with per-deuterated toluene-d8 and nonlabeled toluene than
benzylsuccinate synthase in growing cells exhibited, confirming that
the benzylsuccinate synthase reaction is the major
isotope-fractionating step. This is consistent with previous reports
which showed that isotope fractionation decreases when the supply of
the enzyme with educts is less than saturation (11, 26,
27).
It has been proposed that benzylsuccinate synthase is a glycyl radical
enzyme which abstracts one hydrogen radical from toluene
in a primary
step and returns the identical hydrogen to the benzylsuccinate
molecule
produced (Fig.
7) (
2,
13,
19). To study the benzylsuccinate
synthase mechanism, the
products of the enzyme assay performed
with per-deuterated
toluene-
d8 and nondeuterated toluene in
equal
amounts were analyzed by mass spectroscopy.
Benzylsuccinate-
d8 and nonlabeled
benzylsuccinate were detected, and nondeuterated
benzylsuccinate was
produced 11 times faster than
benzylsuccinate-
d8.
Not even traces of
benzylsuccinate-
d7 and
benzylsuccinate-
d1 masses
were detectable,
which had to be expected if a hydrogen was attached
to the glycyl
radical enzyme before substrate binding and was
exchanged with a
hydrogen atom from benzylsuccinate before product
release. Absence of
these masses derived from transfer of hydrogen
radicals to other
substrate molecules was predicted from the proposed
reaction mechanism
(Fig.
7) (
13). A similar experiment was performed
earlier
with the toluene-degrading, denitrifying strain T and
with
toluene-
d3 and nondeuterated toluene as
the substrates (
2).
However, the hydrogen isotope
fractionation effects described
above were not considered. Unless the
different deuterated toluene
species are turned over completely, the
distribution of benzylsuccinate-
d3 and
benzylsuccinate should be about 90:10 because of the different
turnover
rates. Consequently, the masses of
benzylsuccinate-
d1 and
benzylsuccinate-
d2 that are produced by a
possible alternative
enzyme mechanism must be extremely low. The
distributions of benzylsuccinate
species are evident only if the
different reaction rates of benzylsuccinate
synthase with deuterated
and nondeuterated substrates are known
and taken into account.
View larger version (18K):
[in this window]
[in a new window]
|
FIG. 7.
Reaction mechanism of benzylsuccinate synthase as
proposed by Heider et al. (13). The reaction shown is the reaction in
which per-deuterated toluene-d8 is the
substrate. The deuterium radical subtracted from one molecule of
toluene-d8 by the glycyl radical
enzyme is transferred from the enzyme to the same molecule to
generate benzylsuccinate-d8.
|
|
In this study, we showed that the first enzymatic reaction in anaerobic
toluene degradation is the major process leading to
hydrogen isotope
fractionation. No significant effects of temperature
or changes in the
composition of the bacterial community are expected
as long as anoxic
conditions prevail in the aquifer. Isotope fractionation
during aerobic
degradation of toluene could be influenced by growth
temperatures. The
D/H isotope fractionation during toluene degradation
was 3 orders of
magnitude greater than the
13C/
12C isotope
fractionation for the same bacterial strains described
previously, and
analysis of hydrogen isotope fractionation in
natural environments
might be considered an appropriate tool for
detecting low bacterial
benzene-toluene-ethylbenzene-xylene degradation
activities at
contaminated
sites.
| |
ACKNOWLEDGMENTS |
We thank Matthias Gehre for assistance with the hydrogen isotope analysis.
This work was supported by the Bundesministerium für Bildung und
Forschung (grant 02WT0022) and by the Deutsche Forschungsgemeinschaft (grant Schi 180/7).
| |
FOOTNOTES |
*
Corresponding author. Present address:
Universität Tübingen, Zentrum für Angewandte
Geowissenschaften, Sigwartstr. 10, D-72076 Tübingen, Germany.
Phone: 49-(0)-7071-2976076. Fax: 49-(0)-7071-5059. E-mail:
rainer.meckenstock{at}uni-tuebingen.de.
This paper is publication no. 140 of Deutsche
Forschungsgemeinschaft Priority Program 546 (Geochemical Processes with
Long-Term Effects in Anthropogenically Affected Seepage- and Groundwater).
| |
REFERENCES |
| 1.
|
Ahad, J. M. E.,
B. S. Lollar,
E. A. Edwards,
G. F. Slater, and B. E. Sleep.
2000.
Carbon isotope fractionation during anaerobic biodegradation of toluene: implications for intrinsic bioremediation.
Environ. Sci. Technol.
34:892-896[CrossRef].
|
| 2.
|
Beller, H. R., and A. M. Spormann.
1998.
Analysis of the novel benzylsuccinate synthase reaction for anaerobic toluene activation based on structural studies of the product.
J. Bacteriol.
180:5454-5457[Abstract/Free Full Text].
|
| 3.
|
Beller, H. R., and A. M. Spormann.
1997.
Benzylsuccinate formation as a means of anaerobic toluene activation by the sulfate-reducing strain PRTOL1.
Appl. Environ. Microbiol.
63:3729-3731[Abstract].
|
| 4.
|
Biegert, T.,
G. Fuchs, and J. Heider.
1996.
Evidence that anaerobic oxidation of toluene in the denitrifying bacterium Thauera aromatica is initiated by formation of benzylsuccinate from toluene and fumarate.
Eur. J. Biochem.
238:661-668[Medline].
|
| 5.
|
Blair, N. E., and R. C. Aller.
1995.
Anaerobic methane oxidation on the Amazon shelf.
Geochim. Cosmochim. Acta
59:3707-3715.
|
| 6.
|
Bloom, Y.,
R. Aravena,
D. Hunkeler,
E. Edwards, and S. K. Frape.
2000.
Carbon isotope fractionation during microbial degradation of trichloroethene. cis-1,2-dichloroethene, and vinyl chloride: implications for assessment of natural attenuation.
Environ. Sci. Technol.
34:2768-2772[CrossRef].
|
| 7.
|
Cline, J. D.
1969.
Spectrophotometric determination of hydrogen sulfide in natural waters.
Limnol. Oceanogr.
14:454-458.
|
| 8.
|
Coleman, D. D.,
B. Risatti, and M. Schoell.
1981.
Fractionation of carbon and hydrogen isotopes by methane-oxidizing bacteria.
Geochim. Cosmochim. Acta
45:1033-1037.
|
| 9.
|
Dempster, H.,
B. S. Lollar, and S. Feenstra.
1997.
Tracing organic contaminants in groundwater: a new methodology using compound-specific isotopic analysis.
Environ. Sci. Technol.
31:3193-3197[CrossRef].
|
| 10.
|
Ertl, S.,
F. Seibel,
L. Eichinger,
F. H. Frimmel, and A. Kettrup.
1996.
Determination of the 13C/12C isotope ratio of organic compounds for the biological degradation of tetrachloroethene (PCE) and trichloroethene (TCE).
Acta Hydrochim. Hydrobiol.
24:16-21.
|
| 11.
|
Fogel, M. L., and L. A. Cifuentes.
1993.
Isotope fractionation during primary production, p. 73-97.
In
H. M. Engel, and S. A. Macko (ed.), Organic geochemistry. Plenum Press, New York, N.Y.
|
| 12.
|
Hayes, J. M.,
K. H. Freeman,
B. N. Po, and C. H. Hoham.
1990.
Compound-specific isotope analysis. A novel tool for reconstruction of ancient biogeochemical processes.
Adv. Org. Geochem.
16:1115-1128.
|
| 13.
|
Heider, J.,
A. M. Spormann,
H. R. Beller, and F. Widdel.
1999.
Anaerobic bacterial metabolism of hydrocarbons.
FEMS Microbiol. Rev.
22:459-473[CrossRef].
|
| 14.
|
Hilkert, A. W.,
C. B. Douthitt,
H. J. Schlüter, and W. A. Brand.
1999.
Isotope ratio monitoring gas chromatography/mass spectrometry of D/H by high temperature conversion isotope ratio mass spectrometry.
Rapid Commun. Mass Spectrom.
13:1226-1230[CrossRef][Medline].
|
| 15.
|
Hoefs, J.
1997.
Stable isotope geochemistry, 4th ed.
Springer Verlag, Berlin, Germany.
|
| 16.
|
Hunkeler, D.,
R. Aravena, and B. J. Butler.
1999.
Monitoring microbial dechlorination of tetrachloroethene (PCE) in groundwater using compound-specific stable carbon isotope ratios: microcosm and field studies.
Environ. Sci. Technol.
33:2733-2738[CrossRef].
|
| 17.
|
Krzycki, J. A.,
W. R. Kenealy,
M. J. DeNiro, and J. G. Zeikus.
1987.
Stable isotope fractionation by Methanosarcina barkeri during methanogenesis from acetate, methanol, or carbon dioxide-hydrogen.
Appl. Environ. Microbiol.
53:2597-2599[Abstract/Free Full Text].
|
| 18.
|
Lebedew, W. C.,
W. M. Owsjannikow,
G. A. Mogilewskij, and W. M. Bogdanow.
1969.
Fraktionierung der Kohlenstoffisotope durch mikrobiologische Prozesse in der biochemischen Zone.
Angew. Geol.
15:621-624.
|
| 19.
|
Leuthner, B.,
C. Leutwein,
H. Schultz,
P. Hörth,
W. Haehnel,
E. Schlitz,
H. Schägger, and J. Heider.
1998.
Biochemical and genetic characterization of benzylsuccinate synthase from Thauera aromatica: a new glycyl radical enzyme catalysing the first step in anaerobic toluene metabolism.
Mol. Microbiol.
28:615-628[CrossRef][Medline].
|
| 20.
|
Meckenstock, R. U.
1999.
Fermentative toluene degradation in anaerobic defined syntrophic cocultures.
FEMS Microbiol. Lett.
177:67-73[CrossRef][Medline].
|
| 21.
|
Meckenstock, R. U.,
E. Annweiler,
R. Warthmann,
B. Schink,
W. Michaelis, and H. H. Richnow.
1999.
13C/12C stable isotope fractionation of toluene by anaerobic degradation: a new method to monitor biological degradation in situ?, p. 219-227.
In
R. Fass, Y. Flashner, and S. Reuveny (ed.), Novel approaches for bioremediation of organic pollution. Kluwer Academic Publishers, New York, N.Y.
|
| 22.
|
Meckenstock, R. U.,
B. Morasch,
R. Warthmann,
B. Schink,
E. Annweiler,
W. Michaelis, and H. H. Richnow.
1999.
13C/12C isotope fractionation of aromatic hydrocarbons during microbial degradation.
Environ. Microbiol.
1:409-414[CrossRef][Medline].
|
| 23.
|
Merritt, D. A.,
W. A. Brand, and J. M. Hayes.
1994.
Isotope ratio-monitoring gas chromatography mass spectrometry methods for isotopic calibration.
Org. Geochem.
21:573-583.
|
| 24.
|
Müller, J. A.,
A. S. Galushko,
A. Kappler, and B. Schink.
1999.
Anaerobic degradation of m-cresol by Desulfobacterium cetonicum is initiated by formation of 3-hydroxybenzylsuccinate.
Arch. Microbiol.
172:287-294[CrossRef][Medline].
|
| 25.
|
O'Leary, M. H.
1980.
Determination of heavy-atom isotope effects on enzyme-catalyzed reactions, p. 83-103.
In
D. L. Purich (ed.), Enzyme kinetics and mechanism, vol. 64. Academic Press, New York, N.Y.
|
| 26.
|
O'Neil, J. R.
1986.
Theoretical and experimental aspects of isotopic fractionation, p. 1-37.
In
J. W. Valley, H. P. Taylor, and J. R. O'Neil (ed.), Stable isotopes in high temperature geological processes, vol. 16. Mineralogical Society of America, Washington, D.C.
|
| 27.
|
Peterson, B. L., and B. Fry.
1987.
Stable isotopes in ecosystem studies.
Annu. Rev. Ecol. Syst.
18:293-320[CrossRef].
|
| 28.
|
Rayleigh, J. W. S.
1896.
Theoretical considerations respecting the separation of gases by diffusion and similar processes.
Philos. Mag.
42:493-498.
|
| 29.
|
Richnow, H. H., and R. U. Meckenstock.
1999.
Isotopen-geochemisches Konzept zur in situ Erfassung des biologischen Abbaus in kontaminiertem Grundwasser.
TerraTech.
5:38-41.
|
| 30.
|
Sambrook, J.,
E. F. Fritsch, and R. Maniatis.
1989.
Molecular cloning: a laboratory manual, 2nd ed.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 31.
|
Shaw, J. P., and S. Harayama.
1992.
Purification and characterisation of the NADH:acceptor reductase component of xylene monooxygenase encoded by the TOL plasmid pWW0 of Pseudomonas putida mt-2.
Eur. J. Biochem.
209:51-61[Medline].
|
| 32.
|
Stahl, W. J.
1980.
Compositional changes and 13C/12C fractionations during the degradation of hydrocarbons by bacteria.
Geochim. Cosmochim. Acta
44:1903-1907.
|
| 33.
|
Stookey, L. L.
1970.
Ferrozinea new spectrophotometric reagent for iron.
Anal. Chem.
42:779-781[CrossRef].
|
| 34.
|
Sturchio, N. C.,
J. L. Clausen,
L. J. Heraty,
L. Huang,
B. D. Holt, and T. A. Abrajano.
1998.
Chlorine isotope investigation of natural attenuation of trichloroethene in an aerobic aquifer.
Environ. Sci. Technol.
32:3037-3042[CrossRef].
|
| 35.
|
Ward, J. A. M.,
J. M. E. Ahad,
G. Lacrampe-Couloume,
G. F. Slater,
E. A. Edwards, and B. S. Lollar.
2000.
Hydrogen isotope fractionation during methanogenic degradation of toluene: potential for direct verification of bioremediation.
Environ. Sci. Technol.
34:4577-4581[CrossRef].
|
| 36.
|
Whiticar, M. J., and E. Faber.
1986.
Methane oxidation in sediment and water column environmentsisotope evidence.
Adv. Org. Geochem.
10:759-768.
|
| 37.
|
Widdel, F., and F. Bak.
1992.
Gram-negative mesophilic sulfate-reducing bacteria, p. 3352-3378.
In
A. Balows, H. G. Trüper, M. Dworkin, W. Harder, and K. H. Schleifer (ed.), The prokaryotes, 2nd ed., vol. 4. Springer Verlag, New York, N.Y.
|
| 38.
|
Wilkes, H.,
C. Boreham,
G. Harms,
K. Zengler, and R. Rabus.
2000.
Anaerobic degradation and carbon isotopic fractionation of alkylbenzenes in crude oil by sulphate-reducing bacteria.
Org. Geochem.
31:101-115.
|
| 39.
|
Worsey, M. J., and P. A. Williams.
1975.
Metabolism of toluene and xylenes by Pseudomonas putida (arvilla) mt-2: evidence for a new function of the TOL plasmid.
J. Bacteriol.
124:7-13[Abstract/Free Full Text].
|
Applied and Environmental Microbiology, October 2001, p. 4842-4849, Vol. 67, No. 10
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.10.4842-4849.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Somsamak, P., Richnow, H. H., Haggblom, M. M.
(2006). Carbon Isotope Fractionation during Anaerobic Degradation of Methyl tert-Butyl Ether under Sulfate-Reducing and Methanogenic Conditions. Appl. Environ. Microbiol.
72: 1157-1163
[Abstract]
[Full Text]
-
Nijenhuis, I., Andert, J., Beck, K., Kastner, M., Diekert, G., Richnow, H.-H.
(2005). Stable Isotope Fractionation of Tetrachloroethene during Reductive Dechlorination by Sulfurospirillum multivorans and Desulfitobacterium sp. Strain PCE-S and Abiotic Reactions with Cyanocobalamin. Appl. Environ. Microbiol.
71: 3413-3419
[Abstract]
[Full Text]
-
Morasch, B., Richnow, H. H., Vieth, A., Schink, B., Meckenstock, R. U.
(2004). Stable Isotope Fractionation Caused by Glycyl Radical Enzymes during Bacterial Degradation of Aromatic Compounds. Appl. Environ. Microbiol.
70: 2935-2940
[Abstract]
[Full Text]
-
Mancini, S. A., Ulrich, A. C., Lacrampe-Couloume, G., Sleep, B., Edwards, E. A., Lollar, B. S.
(2003). Carbon and Hydrogen Isotopic Fractionation during Anaerobic Biodegradation of Benzene. Appl. Environ. Microbiol.
69: 191-198
[Abstract]
[Full Text]
-
Manefield, M., Whiteley, A. S., Griffiths, R. I., Bailey, M. J.
(2002). RNA Stable Isotope Probing, a Novel Means of Linking Microbial Community Function to Phylogeny. Appl. Environ. Microbiol.
68: 5367-5373
[Abstract]
[Full Text]
-
Morasch, B., Richnow, H. H., Schink, B., Vieth, A., Meckenstock, R. U.
(2002). Carbon and Hydrogen Stable Isotope Fractionation during Aerobic Bacterial Degradation of Aromatic Hydrocarbons. Appl. Environ. Microbiol.
68: 5191-5194
[Abstract]
[Full Text]
-
Hunkeler, D., Meckenstock, R., Richnow, H. H.
(2002). Quantification of Isotope Fractionation in Experiments with Deuterium-Labeled Substrate. Appl. Environ. Microbiol.
68: 5205-5207
[Full Text]
Top
Abstract
Introduction
