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Applied and Environmental Microbiology, November 2000, p. 4870-4876, Vol. 66, No. 11
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Evidence of Substantial Carbon Isotope
Fractionation among Substrate, Inorganic Carbon, and Biomass during
Aerobic Mineralization of 1,2-Dichloroethane by
Xanthobacter autotrophicus
D.
Hunkeler* and
R.
Aravena
Department of Earth Sciences, University of
Waterloo, Waterloo, Ontario, Canada
Received 26 April 2000/Accepted 1 September 2000
 |
ABSTRACT |
Carbon isotope fractionation during aerobic mineralization of
1,2-dichloroethane (1,2-DCA) by Xanthobacter autotrophicus
GJ10 was investigated. A strong enrichment of 13C in
residual 1,2-DCA was observed, with a mean fractionation factor
± standard deviation of 0.968 ± 0.0013 to 0.973 ± 0.0015. In addition, a large carbon isotope fractionation between
biomass and inorganic carbon occurred. A mechanistic model that links the fractionation factor to the rate constants of the first catabolic enzyme was developed. Based on the model, it was concluded that the strong enrichment of 13C in 1,2-DCA arises because
the first irreversible step of the initial enzymatic transformation of
1,2-DCA consists of an SN2 nucleophilic substitution.
SN2 reactions are accompanied by a large kinetic isotope
effect. The substantial carbon isotope fractionation between biomass
and inorganic carbon could be explained by the kinetic isotope effect
associated with the initial 1,2-DCA transformation and by the metabolic
pathway of 1,2-DCA degradation. Carbon isotope fractionation during
1,2-DCA mineralization leads to 1,2-DCA, inorganic carbon, and biomass
with characteristic carbon isotope compositions, which may be used to
trace the process in contaminated environments.
| |
INTRODUCTION |
1,2-Dichloroethane (1,2-DCA) has
been produced in larger quantities than any other chlorinated
hydrocarbon (35). This compound is mainly used as a
precursor for poly(vinyl chloride) production and as a solvent. 1,2-DCA
is frequently detected in the environment and has been classified as a
priority pollutant by the United States Environmental Protection
Agency. Because of its high aqueous solubility and low sorption
coefficient (37), 1,2-DCA is likely to contaminate
groundwater if it is released into the environment. Under abiotic
conditions, dissolved 1,2-DCA is transformed slowly and toxic products
such as vinyl chloride may be formed (26). In contrast,
microorganisms can rapidly degrade 1,2-DCA to nontoxic end products.
Under anaerobic conditions, ethene is usually the main degradation
product (12), but chloroethane and ethane production has
also been observed (19). Under aerobic conditions, 1,2-DCA is completely mineralized to CO2 and Cl
(15, 24, 25, 40, 45). Anaerobic degradation of 1,2-DCA to
ethene has been observed at several field sites, while aerobic degradation has been reported less frequently (6, 10, 28, 29). Aerobic biodegradation of 1,2-DCA is more difficult to demonstrate at field sites than anaerobic degradation because the end
products of aerobic degradation, inorganic carbon and Cl,
often occur at high background concentrations in groundwater while the
main end product of aerobic degradation, ethene, is less common.
The use of compound-specific isotope analysis is a promising tool for
substantiating intrinsic biodegradation of organic contaminants in
groundwater (1, 21, 47). This method relies on the frequent occurrence during abiotic and biotic transformation processes (2,
39) of a kinetic isotope effect, which consists of differences in
reaction rates for molecules of a compound containing light (12C, H, or 35Cl) and heavy (13C,
D, or 37Cl) isotopes, respectively. As a result, precursor
and products isotope ratios differ; this is called (kinetic) isotope
fractionation. Usually the product is depleted of heavy isotopes
relative to the precursor and, therefore, the precursor becomes
increasingly enriched in heavy isotopes as the reaction proceeds. Large
kinetic isotope effects frequently occur with respect to atoms that
constitute the bond that is broken or formed during the reaction step
(primary isotope effect), while small effects occur with respect to
atoms at other positions (secondary isotope effect). The occurrence of
kinetic isotope effects has been used extensively in organic chemistry
and enzymology to investigate reaction mechanisms and to identify
rate-limiting steps in multistep transformation processes (see, e.g.,
references 9 and 13).
During microbial degradation, an enrichment of heavy isotopes in the
remaining substrate is expected if the initial enzymatic transformation
step is accompanied by a kinetic isotope effect (17). Such
an effect has been observed during reductive dechlorination of
tetrachloroethene, trichloroethene,
cis-1,2-dichloroethene, and vinyl chloride (4,
21, 38) and oxidation of dichloromethane (18). In
contrast, only a small or no carbon isotope fractionation was observed
during aerobic or anaerobic degradation of aromatic hydrocarbons
(27, 31, 38). Isotope analysis can be used not only to
demonstrate that a contaminant is being degraded but also to link
nonunique degradation end products, such as ethene, ethane, and
dissolved inorganic carbon (DIC), to degrading contaminants. Recent
studies (4, 21) have shown that ethene produced by reductive
dechlorination of chlorinated ethenes is initially very depleted in
13C. In contrast, during biodegradation of hydrocarbons,
only a small carbon isotope fractionation between substrate and
inorganic carbon has been observed (14, 16). Analysis of
13C/12C ratios in DIC has been used to
distinguish between inorganic carbon produced by contaminant
degradation and inorganic carbon resulting from carbonate dissolution
(5, 22).
The aim of this study was to investigate and quantify carbon isotope
fractionation among substrate, biomass, and inorganic carbon during
aerobic mineralization of 1,2-DCA by a pure microbial culture. This
study is a first step in the evaluation of the utility of
compound-specific isotope analysis in assessment of 1,2-DCA degradation
at contaminated sites. Several microorganism, in pure cultures, are
known to be capable of aerobic degradation of 1,2-DCA (15, 25, 40,
45). For this study, Xanthobacter autotrophicus GJ10
was chosen since the organism is well characterized (24) and
has previously been used to treat contaminated groundwater (41). Furthermore, the reaction mechanism of the enzyme that catalyzes the initial transformation of 1,2-DCA, haloalkane
dehalogenase, is thoroughly documented (36, 48). In previous
studies of isotope fractionation during biodegradation of organic
contaminants, an empirical model was used to quantify isotope
fractionation (4, 18, 38). In contrast, in this study we
developed a mechanistic model to explain the origin and magnitude of
the observed high degree of isotope fractionation. The use of a
mechanistic model makes it possible to draw some general conclusions
with regard to the occurrence and characteristics of isotope
fractionation during contaminant degradation.
| |
MATERIALS AND METHODS |
Organism and growth conditions.
X. autotrophicus GJ10
was obtained from D. B. Janssen (Department of Biochemistry,
University of Groningen, Groningen, The Netherlands). The organism,
originally isolated from a mixture of activated sludge and chemically
polluted soils, is capable of growing on 1,2-DCA as a sole carbon and
energy source (25). It constitutively produces two different
dehalogenases (24): one is specific for halogenated alkanes,
while the other is specific for halogenated carboxylic acids. In the
initial step, which may cause enrichment of heavy isotopes in the
residual substrate, 1,2-DCA is transformed to 2-chloroethanol by
hydrolytic dehalogenation (24).
The mineral medium employed was similar to the one used by Janssen and
coworkers (25) except that the strength of the phosphate buffer was reduced since lower 1,2-DCA concentrations were used. The
medium, which contained (per liter) 3.22 g of
Na2HPO4 · 12H2O, 0.81 g
of KH2PO4, 0.5 g of
(NH4)2SO4, 0.2 g of
MgSO4 · 7H2O, and 0.015 g of
CaCl2 · 2H2O, was supplemented with 1 ml
of trace element solution (42) per liter and adjusted to pH
7.2 before being autoclaved. Afterward, 0.1 ml of a sterile vitamin
solution (44) was added per liter of medium. The culture was
grown in 250-ml glass bottles which contained 185 ml of medium and were
closed with Mininert-Valves (Vici Precision Sampling, Baton Rouge,
La.). Before the experiments were begun, the culture was transferred three times. Each subculture received 0.61 mM (60 ppm) of 1,2-DCA at
four time points.
Experiments.
For all experiments, 15 ml of the previous
subculture was added to bottles containing 170 ml of autoclaved medium.
To remove DIC, the medium was purged for 2 h with helium by the
use of a sterile stainless steel needle. Afterward, 20 ml of
O2 and 1,2-DCA (99.8% purity; Aldrich, Milwaukee, Wis.)
were added and the bottles were placed on a shaker. After 1 h of
shaking, samples representing initial concentrations were removed.
In experiment I, 1,2-DCA was added at a concentration of 0.61 mM (60 ppm). The cultures were incubated in duplicate at 10
and 23°C,
respectively, and continuously shaken. The concentration
and
13C/
12C ratio of 1,2-DCA were determined at
various time points during
degradation. Furthermore, the concentrations
and
13C/
12C ratios of inorganic carbon and
biomass were determined at the
beginning and the end of the
experiment.
In experiment II, 1,2-DCA was added at a concentration of 1.22 mM (120 ppm). A higher concentration than that used in experiment
I was chosen
in order to obtain inorganic carbon and biomass at
concentrations
sufficient to determine
13C/
12C ratios at
intermediate stages. Six bottles were prepared and
incubated at 23°C.
The concentration and
13C/
12C ratio of 1,2-DCA
in one of the cultures were determined at various
times during
degradation. The other cultures were stopped at different
time points
by adding HgCl
2 (25 ppm Hg
2+), and the
concentrations and
13C/
12C ratios of 1,2-DCA,
inorganic carbon, and biomass were measured.
In addition, the
Cl
concentration was measured at the beginning and the
end of the
experiment to confirm that 1,2-DCA mineralization had
reached
completion.
Sampling.
For concentration and
13C/12C ratio analyses of 1,2-DCA, liquid
samples (2-ml volume) were taken and preserved with HgCl2
(25 ppm of Hg2+). To determine the concentration and
13C/12C ratio of CO2, 0.5 ml of
headspace gas was removed and analyzed immediately. For concentration
and 13C/12C ratio analyses of DIC, 2.5-ml
volumes of liquid sample were dispensed into helium-filled 10-ml
Vacutainer tubes (Becton Dickinson, Franklin Lakes, N.J.), displacing
the same volume of helium. Immediately after the sampling, 0.1 ml of
100% H3PO4 was added to each sample to stop
biological processes and to transform all dissolved inorganic carbonate
to carbonic acid and CO2. To determine optical densities, 1-ml volumes of liquid samples were removed. To analyze chloride concentrations, 2-ml volumes of liquid samples were taken and preserved
with sodium azide. In all cases, the removed sample was replaced by a
similar volume of helium. The biomass was collected by centrifugation
at 12,000 × g for 10 min. For analysis of biomass in
intermediate samples, the culture was inactivated by adding HgCl2 (25 ppm of Hg2+) and purged for 3 h
with helium to remove volatile compounds before centrifugation. The
precipitate was washed twice with 15 mM phosphate buffer and
freeze-dried.
Analytical methods.
All stable-isotope analyses were
performed in the Environmental Isotope Laboratory of the University of
Waterloo. 13C/12C ratios are reported in the
usual delta notation (
13C). The 13C value
is defined as 13C = (Rs/Rr 
1) × 1,000, where
Rs and Rr are the
13C/12C ratios of the sample and the
international standard Vienna Peedee Belemnite (VPDB), respectively.
The
13C of pure-phase 1,2-DCA was determined as
previously described (
20). The
13C values of
dissolved 1,2-DCA and headspace CO
2 were determined
with a
gas chromatography-combustion-isotope ratio mass spectrometry
(GC-C-IRMS) system. The GC-C-IRMS system consisted of an Agilent
(Palo
Alto, Calif.) gas chromatograph equipped with a split/splitless
injector, a Micromass (Manchester, United Kingdom) combustion
interface
operated at 850°C, and a Micromass Isochrom isotope-ratio
mass
spectrometer. For headspace analysis of CO
2, the gas
chromatograph
was equipped with a GS-GasPro column (J&W Scientific,
Folsom,
Calif.). The
13C values of dissolved 1,2-DCA
were determined by a method based
on solid-phase microextraction as
described by Hunkeler and Aravena
(
20). Concentrations of
CO
2 and 1,2-DCA were determined based
on the peak areas of
the mass 44 signal for samples and external
standards. The analytical
system was verified daily by using reference
compounds with known
13C values (
20). The standard uncertainty of
the
13C measurement was ± 0.5
for
CO
2 and 1,2-DCA (
n = 3). The relative
standard uncertainty of the concentration measurement was ± 8%
for CO
2 and 1,2-DCA (
n = 3).
Carbon isotope ratios and concentrations of DIC were determined with a
µGas breath analyzer coupled to a Micromass Isochrom
isotope-ratio
mass spectrometer (Micromass). External DIC standards
were prepared by
dissolution of NaHCO
3 with a known
13C in
distilled water that had been purged with helium for 0.5
h.
Standards and samples were shaken for at least 1 h prior to
carbon
isotope analysis. DIC concentrations were quantified based
on the peak
areas of the mass 44-ion trace of samples and external
standards. The
standard uncertainty of the
13C measurement was ± 0.15
for DIC (
n = 6). The relative standard
uncertainty of the concentration measurement was ± 4% for DIC
(
n = 6).
Carbon isotope ratios and carbon content of biomass were determined
with a CE Instruments (Rodano, Italy) elemental analyzer
coupled to a
Micromass Isochrom isotope-ratio mass spectrometer.
Samples of 1 mg
were packed into tin cups and combusted at 1,030°C.
The standard
uncertainty of the
13C of biomass was ± 0.6
,
while the relative standard uncertainty
of the carbon content was ± 10% (
n = 4).
Chloride concentrations were analyzed with a Dionex (Sunnyvale, Calif.)
ion chromatography system equipped with an AS4A-SC
column (Dionex).
Optical densities were determined at 600 nm using
an Ultraspec Plus
UV/visible spectrophotometer (Amersham Pharmacia
Biotech).
Calculations.
For 1,2-DCA and inorganic carbon, expected
concentrations if the total masses of these compounds were present in
the aqueous phase are reported. This allows for easy comparison of
1,2-DCA consumption and production of inorganic carbon, biomass, and
Cl. For 1,2-DCA, 13C values determined by
solid-phase microextraction of the aqueous phase are given. They
correspond to the 13C of the total mass of 1,2-DCA since
only a very small carbon isotope fractionation between 1,2-DCA in the
aqueous phase and headspace occurs (20) and since the mass
of 1,2-DCA in the headspace is small. In contrast, a significant
pH-dependent carbon isotope fractionation occurs between headspace
CO2 and DIC. At pH 7.2, CO2 in the headspace is
depleted of 13C by 7 compared to DIC (8).
Therefore, the 13C of the total inorganic carbon was
calculated based on concentrations and 13C values of DIC
in the aqueous phase and CO2 in the headspace. The biomass
concentration was calculated by multiplying the carbon content by the
total mass of freeze-dried biomass and dividing the result by the
volume of the aqueous phase. The 13C of the produced
biomass was calculated based on the concentrations and
13C values of biomass at the beginning of the experiment
and at the time of sampling. The 13C-mass balance was
calculated by multiplying the concentrations of 1,2-DCA, inorganic
carbon, and biomass by their 13C values, adding the
contributions, and dividing the sum by the total concentration of these compounds.
Isotope fractionation during biodegradation of 1,2-DCA was quantified
using a Rayleigh-type evolution model (
7,
8,
30).
According
to this model, the isotopic composition of the substrate
is given by
|
(1)
|
where
is the fraction of substrate remaining,
RS is the substrate isotope ratio at a remaining
fraction
,
RS0 is the initial
isotope ratio of the substrate, and
is the fractionation
factor.
The fractionation factor (
30) is defined by
|
(2)
|
where
dP13 and
dP12 are increments of product containing
13C and
12C, respectively, which appear in an
infinitely short period of
time (instantaneous product) and
S13 and
S12 are the
concentrations
of substrate with
13C and
12C,
respectively.
By using the
13C notation for carbon isotope ratios,
equation 1 transforms to
|
(3)
|
where
13C
S is the carbon
isotope ratio of the substrate at a remaining fraction
and
13C
S0 is the initial
carbon isotope ratio of the substrate.
The fractionation factor
was
quantified on the basis of equation
3 by linear
regression.
| |
RESULTS |
Concentrations and carbon isotope ratios. (i) Experiment I.
At
23°C, 1,2-DCA (60 ppm) was consumed by X. autotrophicus
GJ10 within about 20 h and the optical density increased (Fig. 1a). The 13C of added
1,2-DCA (initial 13C, 30.6) increased to values of
more than +10 during the experiment. Similar concentrations and
13C values were obtained for duplicate batch cultures.
The produced inorganic carbon was depleted in 13C
(13C = 46.2) and the biomass was enriched in
13C (13C = 17.2) compared to the
initially added 1,2-DCA (13C = 30.6). Degradation
of 1,2-DCA took about four times longer at 10°C than at 23°C.
Again, a strong increase in the 13C of 1,2-DCA was
observed during its biodegradation (Fig. 1b).
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FIG. 1.
Experiment I: relative concentration of 1,2-DCA (closed
circles, top panel), 13C of 1,2-DCA (closed circles,
bottom panel), and optical density (closed squares) in batch cultures
of X. autotrophicus GJ10 at 23°C (a) and 10°C (b). The
initial concentration of 1,2-DCA was 60 ppm.
|
|
(ii) Experiment II.
In experiment II, 120 ppm of 1,2-DCA was
consumed within about 30 h and the optical density increased (Fig.
2). The amount of inorganic carbon
relative to the carbon initially added as 1,2-DCA reached a final value
of 0.4 (Fig. 2). The 13C of 1,2-DCA increased from
30.6 to +28.0. The 13C of inorganic carbon in the
continuous microcosm was 78.0 in the first intermediate sample and
increased to a final value of 47.4 (Fig. 2). Similar
concentrations and 13C values were obtained for
continuous and sacrificial microcosms at similar points in time (Fig.
2). The yield of inorganic carbon (inorganic carbon production divided
by 1,2-DCA consumption in millimolar concentration of C) remained
nearly constant throughout the experiment, with an average of 0.42 (Table 1). The 13C of
produced biomass was 44.2 in the first intermediate sample and
increased to 18.3 (Table 1; Fig. 2). The concentration and
13C of the sum of 1,2-DCA, inorganic carbon, and biomass
in intermediate and final samples corresponded to the initial
concentration and 13C of 1,2-DCA (Table 1). This
indicates that all products containing carbon were recovered and that
accumulation of intermediate degradation products was negligible. The
total production of chloride was 2.45 mM, which confirmed that complete
degradation of 1,2-DCA took place.
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FIG. 2.
Experiment II: relative concentration of 1,2-DCA (closed
circles, top panel), yield of DIC (closed inverted triangles, top
panel), 13C of 1,2-DCA (closed circles, bottom panel),
13C of inorganic carbon (closed inverted triangles,
bottom panel), and optical density (closed squares) in batch culture of
X. autotrophicus GJ10 incubated at 23°C. Open symbols are
corresponding values in sacrificial batch cultures. The initial
concentration of 1,2-DCA was 120 ppm. Open diamond, 13C
of biomass in sacrificial batch cultures; dashed line,
13C of added 1,2-DCA.
|
|
Fractionation factors.
To be able to compare the magnitudes of
isotope fractionation during the different experiments, fractionation
factors were calculated based on equation 3. The regression
coefficients are close to 1 (
0.9878), indicating that the
fractionation factor remained essentially constant throughout the
experiments (Table 2). Fractionation
factors of duplicate experiments agreed closely, and only small
differences were observed for different initial concentrations and
temperature.
| |
DISCUSSION |
Aerobic mineralization of 1,2-DCA by X. autotrophicus
GJ10 is accompanied by a strong carbon isotope fractionation. The
obtained fractionation factors are the largest yet reported for
biodegradation of two-carbon organic compounds. Significant carbon
isotope fractionation has previously been observed during reductive
dechlorination of trichloroethene (0.993 to 0.998 [4, 21,
38]), cis-1,2-dichloroethene (0.985 to 0.988 [4, 21]), and vinyl chloride (0.974 to 0.975 [4, 21]) and during aerobic oxidation of
dichloromethane (0.958 [18]). In contrast, during both
aerobic and anaerobic degradation of toluene, only a small carbon
isotope fractionation occurred (0.997 to 0.998 [31]).
As shown in the present study, aerobic oxidation of 1,2-DCA is
accompanied not only by a strong enrichment of 13C in
1,2-DCA but also by an unusually large fractionation of carbon isotopes
between inorganic carbon and biomass.
Carbon isotope fractionation during initial transformation.
The magnitude of the fractionation factor depends on the magnitude
of the isotope fractionation occurring during transport of 1,2-DCA
across the cell membrane, the magnitude of the isotope fractionation
during its initial transformation, and the relative rates of these two
processes. Since the cell membrane of X. autotrophicus GJ10
does not act as a barrier to permeation of 1,2-DCA (46), it
can be concluded that the observed fractionation factor mainly reflects
isotope fractionation during initial 1,2-DCA transformation. This is
consistent with the observation that the magnitude of the fractionation
factor can be explained only by the action of an enzymatic process
and not by a transport process. Since the catalytic mechanism of the
haloalkane dehalogenase is well understood, it is possible to evaluate
in more detail the origin of the large isotope fractionation during the
initial transformation of 1,2-DCA. Using basic equations of enzyme
kinetics, the fractionation factor can be linked to parameters that
characterize the kinetics of the haloalkane dehalogenase. If substrate
with 12C and 13C at the reactive center is
simultaneously available, as in this study, the amount of product with
12C, d12P, that is formed during an infinitely
short time period dt is given by (39, 43)
|
(4)
|
where
V12 is the limiting rate for
substrate with
12C at the reactive center,
S12 and
S13 are the
concentrations of substrate
with
12C and
13C,
respectively, at the reactive center, and
Km12 and
Km
are the Michaelis constants for substrate with
12C and
13C, respectively, at the reactive
center.
By inserting equation 4 and an analogous equation for
dP13 into equation 2, the following equation for
is obtained:
|
(5)
|
where
13(
V/K), defined by
is the isotope effect on
V/K (
32). Thus,
biodegradation experiments with isotope ratios at natural abundance
levels reflect
isotope effects on
V/K, analogous to enzyme
studies under competitive
conditions (labeled and unlabeled substrate
simultaneously present
[
33]). For the haloalkane
dehalogenase (Fig.
3),
V/K is
given
by
|
(6)
|
where
k1 and
k1
are the rates of formation and dissociation, respectively, of the
substrate enzyme complex,
k2 is
the rate of the
first irreversible catalytic step, and
Et is the
total enzyme concentration. Isotope fractionation during formation
and
dissociation of the substrate-enzyme complex is small compared
to that
during irreversible transformation steps and can usually
be neglected
(
34). Under this assumption, the following expression
for
in terms of the kinetic constants of the enzyme is obtained
by
inserting equation 6 into equation 5:
|
(7)
|
where
C =
12 k2/
k1;
C is
usually referred to as commitment to catalysis since it represents the
tendency of the enzyme-substrate
complex to go forward through
catalysis rather than to break down
to free enzyme and substrate
(
32). The ratio
12k2/
13k2
is usually denoted as the intrinsic isotope effect since it
represents
the full isotope effect originating from a single reaction
step
(
32). Several important conclusions can be drawn from
equation
7. First, the fractionation factor
is expected to be
constant
since the right-hand side of equation 7 contains only rate
constants.
Thus, equation 7 justifies the hypothesis that
is
constant,
which has been postulated in this and in previous studies
(
4).
Second, the magnitude of
or
13(
V/K) depends only on the steps up to and
including the first
irreversible step, as previously observed for
isotope effects
in enzyme studies under competitive conditions
(
34,
39). It
does not depend on whether the rate-limiting
step is accompanied
by isotope fractionation. Indeed, for the
haloalkane dehalogenase,
the slowest step is the release of the
Cl
(Fig.
3), which is not accompanied by carbon isotope
fractionation.
Third, the degree to which the intrinsic isotope effect
(
12k2/
13k2)
is detectable depends on the commitment to catalysis. The
larger the
rate of catalysis (
12k2) is,
compared to the rate of dissociation of the substrate-enzyme
complex
(
k1), and thus the larger
C is, the
smaller is
13(
V/C) for a given value of the
intrinsic isotope effect (equation
7). The equations given above are
specific for the case discussed
in this study. For other enzyme
mechanisms (e.g., two substrates),
a different expression for
C is obtained (
33). Furthermore,
if the rate of
substrate transformation by the first catabolic
enzyme is high compared
to the rate of substrate export from the
cell, a more complex
relationship between
and
13(
V/C) arises.
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FIG. 3.
Reaction scheme for dehaloalkane dehalogenase. The rate
constants have the following values (means ± standard
deviations): k1 = 9 × 103 ± 1 × 103 µM1
s1, k1 = 20 ± 5 s1, k2 = 50 ± 10 s1, k3 = 14 ± 3 s1, and k4 = 8 ± 2 s1 (36). C = k2/k1 = 2.5.
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Based on equation 7, it is possible to relate the magnitude of the
fractionation factor
to the intrinsic isotope effect
associated
with the first irreversible step of 1,2-DCA transformation.
For the
haloalkane dehalogenase, the first irreversible step consists
of an
S
N2 nucleophilic substitution ((
48)), in which a
carboxylate
group of a side chain of the enzyme acts as a nucleophile
and
displaces a chloride ion. From numerous studies in organic
chemistry,
it is known that S
N2 processes are prone to a
large kinetic isotope
effect with
12k/
13k ratios of up to
1.080 (
13). In the present study, an average
fractionation
factor of 0.970 was observed, which was measured
with respect to both
carbon positions of 1,2-DCA. However, in
the first irreversible step
(S
N2 reaction), only one of the two
carbon atoms is
involved in the reaction mechanism. Assuming that
no significant
secondary isotope effect occurs, which is a reasonable
assumption for
carbon isotopes (
23), the fractionation factor
with respect
to the reactive carbon is 0.940. Using this value
and the known value
of
C (2.5 [Fig.
3]), the magnitude of the
intrinsic
isotope effect
(
12k2/
13k2)
for the S
N2 step of the enzyme can be estimated using
equation
7. A value of 1.090 is obtained, which is in the range typical
for S
N2 reactions. In summary, the large carbon isotope
effect
associated with 1,2-DCA transformation by
X. autotrophicus GJ10
can be explained by a combination of (i) a
large kinetic isotope
effect during the first irreversible step of the
initial enzymatic
transformation (S
N2 reaction), (ii) a
relatively small commitment
to catalysis leading to a large
13(
V/K), and (iii) rapid transport of 1,2-DCA
into and out of the
cell, which makes isotope fractionation detectable
outside the
cell.
Carbon isotope fractionation between inorganic carbon and
biomass.
In this study, the inorganic carbon is depleted in
13C by 29.1 to 37.8 compared to the biomass (Table 1).
In previous studies with different substrates, a similar, though much
smaller, 13C depletion in inorganic carbon has been
observed. Inorganic carbon produced by biodegradation of glucose was
depleted in 13C by 2.8 (3), while inorganic
carbon from phenol and benzoate mineralization was depleted by up to
8 (16). The unusually large carbon isotope fractionation
between biomass and inorganic carbon during 1,2-DCA mineralization can
be explained based on the metabolic pathway. 1,2-DCA is transformed via
2-chloroethanol, 2-chloroaldehyde, and chloroacetate to glycolate (Fig.
4), which enters the central metabolic
pathway (24). Glycolate is usually oxidized to glyoxylate
and transformed to acetyl coenzyme A (acetyl-CoA) via the glycerate
pathway by bacteria. Two of the reactions on the way to acetyl-CoA, the
transformation of two glycoxylate molecules to tartronate semialdehyde
and the transformation of pyruvate to acetyl-CoA, involve a
decarboxylation step (Fig. 4). In both of them, the liberated
CO2 molecule originates from the carboxyl position of the
glycolate (the carbon boldfaced in Fig. 4). In contrast, the carbon in
the acetyl-CoA corresponds to the hydroxyl carbon of the glycolate.
Given a position-specific of 0.940 for the initial 1,2-DCA
transformation, the carboxyl carbon is expected to be depleted in
13C by approximately 60 relative to the substrate while
the hydroxyl carbon has a 13C similar to that of the
concurrent substrate. The 13C of the inorganic carbon
depends on the relative amount of inorganic carbon originating from the
two decarboxylation steps on the way to and from acetyl-CoA,
respectively. In Fig. 5, the calculated average 13C of glycolate and the average
13C of its carboxyl-carbon position are plotted and
compared to the 13C of the inorganic carbon. If all
inorganic carbon originated from the carboxyl-carbon position, the
13C of the inorganic carbon would correspond to that of
the carboxyl position of the glycolate. If carboxyl and hydroxyl
positions equally contributed to inorganic carbon production, the
13C of the inorganic carbon should correspond to that of
the glycolate. Figure 5 shows that the 13C of the
inorganic carbon lies between the two calculated curves, indicating
that more inorganic carbon originates from the carboxyl carbon than
from the hydroxyl carbon. Correspondingly, a larger fraction of
isotopically heavier hydroxyl carbon is incorporated into biomass via
acetyl-CoA, glycolate, and/or pyruvate, which leads to an enrichment of
13C in biomass compared to inorganic carbon. In addition to
this effect associated with position-specific 13C
differences in glycolate, isotope fractionation at branch points of the
intracellular carbon flow may also contribute to differences between
biomass and inorganic carbon 13C values (17).
Such an effect is known to occur at the branch point downgradient of
pyruvate (11). However, this effect alone cannot explain the
large isotope fractionation between biomass and inorganic carbon
observed in this study.
View larger version (14K):
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|
FIG. 5.
Experiment II: measured 13C of 1,2-DCA
(closed circles), measured 13C of inorganic carbon
(closed inverted triangles), calculated average 13C of
glycolate (open squares), and calculated average 13C of
carboxyl-carbon position of glycolate (open triangles).
|
|
Summary and conclusions.
A very large kinetic isotope effect
accompanied by an unusually strong carbon isotope fractionation between
biomass and inorganic carbon, was observed during aerobic
transformation of 1,2-DCA by X. autotrophicus GJ10. These
effects led to very characteristic 13C values for
1,2-DCA and inorganic carbon during biodegradation of 1,2-DCA which
potentially could be used to monitor intrinsic biodegradation of this
compound in the environment. The origin and magnitude of the large
kinetic isotope effect during initial substrate transformation were
substantiated by using a mechanistic model. The model makes it possible
to draw some conclusions concerning the occurrence and predictability
of kinetic isotope effects during biodegradation of contaminants. A
prerequisite for the occurrence of a detectable carbon isotope
fractionation during biodegradation of organic compounds is the
occurrence of a significant intrinsic isotope effect during the first
irreversible step of the first enzymatic transformation. Whether this
effect is detectable depends on several factors, including (i) the
number of carbon atoms in the molecule, since the intrinsic isotope
effect usually occurs with respect to a specific position in the
molecule while the isotope ratio measurement is usually compound
specific; (ii) the magnitude of the commitment to catalysis; and (iii)
the rate of transport of the substrate into and out of the cell. The
large number of factors that affect the magnitude of the observed
isotope fractionation makes it difficult to predict isotope
fractionation. Not only the degradation mechanism but also the relative
rates of the various processes must be known. To determine the effect of factor ii, the rate constants of the enzyme have to be known. The
effect of factor iii could be evaluated by comparing isotope fractionation by whole cells with isotope fractionation by cell extracts. Factors i and iii may explain why a large isotope
fractionation has been observed for small molecules, as in this study,
while only a small isotope effect occurs during degradation of larger molecules. In the context of intrinsic biodegradation, the fate of
small mobile molecules is commonly of particular concern. For such
molecules, a detectable isotope fractionation is more likely to occur
and may be a valuable tool to demonstrate intrinsic biodegradation, as
suggested by the first field trials (21). For practical
application of the method, more information about variations of
fractionation factors between different cultures and under different
environmental conditions is required.
| |
ACKNOWLEDGMENTS |
This project was supported by grants from the National Sciences
and Engineering Research Council of Canada, the Center for Research in
Earth and Space Technology, and the University Consortium Solvents-in-Groundwater Research Program.
We thank D. B. Janssen for providing samples of X. autotrophicus GJ10, W. Mark for support during isotope ratio
measurements, and B. Butler for helpful discussions.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Earth Sciences, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1. Phone: (519) 885 1211. Fax: (519) 746 7484. E-mail:
dhunkele{at}sciborg.uwaterloo.ca.
| |
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Abstract
Introduction
