Previous Article | Next Article 
Appl Environ Microbiol, January 1998, p. 208-215, Vol. 64, No. 1
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Effect of Trichloroethylene on the Competitive
Behavior of Toluene-Degrading Bacteria
Astrid E.
Mars,
Gjalt T.
Prins,
Pieter
Wietzes,
Wim
de Koning, and
Dick B.
Janssen*
Department of Biochemistry, University of
Groningen, 9747 AG Groningen, The Netherlands
Received 18 July 1997/Accepted 22 October 1997
 |
ABSTRACT |
The influence of trichloroethylene (TCE) on a mixed culture of four
different toluene-degrading bacterial strains (Pseudomonas putida mt-2, P. putida F1, P. putida
GJ31, and Burkholderia cepacia G4) was studied with a
fed-batch culture. The strains were competing for toluene, which was
added at a very low rate (31 nmol mg of cells [dry
weight]
1 h1). All four strains were
maintained in the mixed culture at comparable numbers when TCE was
absent. After the start of the addition of TCE, the viabilities of
B. cepacia G4 and P. putida F1 and GJ31 decreased 50- to 1,000-fold in 1 month. These bacteria can degrade TCE,
although at considerably different rates. P. putida mt-2, which did not degrade TCE, became the dominant organism. Kinetic analysis showed that the presence of TCE caused up to a ninefold reduction in the affinity for toluene of the three disappearing strains, indicating that inhibition of toluene degradation by TCE
occurred. While P. putida mt-2 took over the culture,
mutants of this strain which could no longer grow on
p-xylene arose. Most of them had less or no
meta-cleavage activity and were able to grow on toluene
with a higher growth rate. The results indicate that cometabolic
degradation of TCE has a negative effect on the maintenance and
competitive behavior of toluene-utilizing organisms that transform TCE.
| |
INTRODUCTION |
A limited number of chlorinated
aliphatic hydrocarbons can support bacterial growth by serving as a
source of carbon and energy (17). In these cases, treatment
of polluted sites or waste streams can be performed by using systems in
which the number of desirable microorganisms increases because they
proliferate at the expense of the contaminants. However, many
chlorinated compounds are biodegradable only by cometabolic conversion.
In this case, the xenobiotic compound does not cause a selective
increase in the population of the active microorganisms. A primary
substrate must be present for growth and maintenance, which, however,
does not necessarily select for the desired degradative activity.
Besides, conversion of the cosubstrate could lead to toxic products
which inhibit the most active organisms.
The cometabolic conversion of trichloroethylene (TCE) by nonspecific
oxygenases of aerobic bacteria is an example of a process which may
harm the bacteria that execute the degradation reaction. The reaction
uses reducing equivalents (9), and TCE can be a competitive
inhibitor of the conversion of the primary growth substrate, which is
usually also required to induce the oxidative enzyme. Furthermore, TCE
conversion can be accompanied by toxic effects; i.e., cell damage may
occur due to nonspecific reactions of TCE conversion products with cell
components, as was shown with cultures of Pseudomonas putida
F1 and Methylosinus trichosporium OB3b (27, 35).
These toxic effects can lead to a large increase in maintenance energy
demand, as was observed in a nongrowing, toluene-limited, fed-batch
culture of Burkholderia cepacia G4, which converted TCE
during cultivation on toluene as the primary substrate (22).
However, despite these negative effects, cometabolic conversion is the
only possibility for aerobic biodegradation of TCE and deserves
significant attention, since no organisms which can grow on this
compound are known.
When organisms are applied for cometabolic TCE degradation during in
situ bioremediation processes, they might frequently face situations
with very low substrate concentrations. Also, in reactors employing
biofilms, the amount of growth substrate that is added is usually kept
as low as possible to prevent the formation of excess biomass. However,
under energy-limiting conditions, negative effects of TCE conversion,
such as an increased maintenance coefficient, could give TCE degraders
a large disadvantage compared to organisms that also use the primary
substrate but do not degrade TCE. This could limit the long-term
stability of a treatment process and lead to reduced conversion rates.
The purpose of the work described in this paper was to determine the
competitiveness of TCE-degrading bacteria at very low primary substrate
concentrations in the absence and presence of TCE. Toluene was taken as
a model primary substrate.
Toluene degradation can start with oxidation of the methyl group or by
direct oxidation of the aromatic ring. The first route is used by
P. putida mt-2, which converts toluene via benzylalcohol, benzaldehyde, and benzoate to catechol (1). Direct oxidation of the aromatic ring is performed by, for example, B. cepacia G4 (25, 26) and P. putida F1
(34). B. cepacia G4 converts toluene via
o-cresol to 3-methylcatechol (3MC) by two subsequent monooxygenase steps (32). P. putida F1 uses a
dioxygenase to convert toluene to toluene dihydrodiol, which is
subsequently oxidized to 3MC (34).
To study the effect of TCE on the competition for small amounts of
toluene between microorganisms, four different toluene degraders were
cultivated together in a fed-batch culture, which allows the use of
very low toluene concentrations (5). Two of the strains
(B. cepacia G4 and P. putida F1) are well known for their ability to cometabolically convert TCE when toluene is the
primary substrate. P. putida mt-2 is unable to degrade TCE,
while P. putida GJ31 (23) converts TCE slightly.
The results show that when TCE was added to the culture, P. putida mt-2 and mutants thereof became the dominant organisms,
which caused a loss of the TCE degradation capacity.
| |
MATERIALS AND METHODS |
Nomenclature.
The following parameters are used in this
paper: ad, specific decay rate
(minute1); C, substrate concentration
(micromolar); H, dimensionless Henry's coefficient;
kLa, mass transfer rate coefficient
(minute1); Ki, inhibition constant
(micromolar); Km, Michaelis-Menten constant
(micromolar); Ks, Monod constant (micromolar);
µmax, maximal specific growth rate
(minute1); rmax, maximal specific
substrate conversion rate (micromoles milligram of cells [dry
weight]1 minute1); t, time
(minutes); V, volume (liters); X, concentration
of biomass (milligrams of cells [dry weight] liter1);
and Y, growth yield (milligrams of cells [dry weight]
micromole1). Subscripts denote the following parameters:
g, gas phase; l, liquid phase; and 0, time zero.
Bacterial strains.
B. cepacia G4 (26) was a
gift from M. S. Shields (U.S. Environmental Protection Agency,
Gulf Breeze, Fla.). P. putida F1 (34) was
provided by L. P. Newman (University of Minnesota, St. Paul).
P. putida GJ31 was previously characterized (23), and P. putida mt-2 (1) was a gift from W. Duetz
(RIVM, Bilthoven, The Netherlands).
Culture conditions.
The organisms were grown in a fed-batch
fermentor with a working volume of 2.5 liters. The mineral medium (MMV)
contained (per liter) 5.3 g of
Na2HPO4 · 12H2O, 1.4 g
of KH2PO4, 0.2 g of MgSO4
· 7H2O, 1.0 g of
(NH4)2SO4, 1 ml of a vitamin
solution (16), and 5 ml of a trace element solution. The
trace element solution contained (per liter) 780 mg of
Ca(NO3)2 · 4H2O, 200 mg of
FeSO4 · 7H2O, 10 mg of
ZnSO4 · 7H2O, 10 mg of
H3BO3, 10 mg of CoCl2 · 6H2O, 10 mg of CuSO4 · 5H2O,
4 mg of MnSO4 · H2O, 3 mg of
Na2MoO4 · 2H2O, 2 mg of
NiCl2 · 6H2O, and 2 mg of
Na2WO4 · 2H2O. Each component of the medium was autoclaved separately, except the phosphates and the trace elements, which were autoclaved together, and
the vitamin solution, which was filter sterilized prior to addition.
The pH was adjusted to 7.2 with autoclaved 0.5 M NaOH and 0.25 M
H2SO4. The temperature was set at 28°C, and
the impeller speed was set at 1,500 rpm.
Toluene was supplied to the culture via the gas phase. This was done by
leading an airflow via a glass filter (P3; Elgebe, Leek, The
Netherlands) through ice-cold toluene prior to addition to the culture.
TCE was added via the gas phase from a gas cylinder containing 475 ppm
of TCE in air (AGA Gas BV, Amsterdam, The Netherlands). Extra
water-saturated air was added to the culture to supply sufficient oxygen. The flow rates of the gases are given in Table
1. All gas flows were filter sterilized
before addition to the culture. Flows were controlled with mass flow
controllers (type F201C-FA-11-V; 0 to 5 ml min1, 0 to 20 ml min1, and 0 to 500 ml min1; Bronkhorst
High-Tec B.V., Veenendaal, The Netherlands). The outgoing gas stream
was led through a water column under slight overpressure, which
facilitated the detection of possible leakage.
Analysis of the culture.
The culture density of each sample
was estimated by measuring the optical density at 450 nm
(OD450) on a Hitachi 100-60 spectrophotometer and by
determining the protein concentration as described by Lowry et al.
(21) with bovine serum albumin as a standard. The dry weight
of the culture was determined after 40 days by centrifuging duplicate
100-ml samples of culture (15 min, 6,000 × g, 4°C), washing the pellets with the same volume of cold demineralized water,
and drying the pellets to a constant weight in a preweighed aluminum
cup for 2 to 3 days at 80°C.
The viability of the culture was determined by diluting a culture
sample and counting the number of colonies (CFU) after the
sample was
plated on 1.5% agar plates containing either rich medium
(nutrient
broth [NB] plates) or mineral medium to which 10 mg
of yeast extract
liter
1 was added (MMY) instead of the vitamin solution.
One day after
plating, 5 to 10 µl of pure toluene (Tol plates) was
added on
a paper filter disk in the cover of the petri dish. This was
done
to prevent the immediate exposure of the cells to high toluene
concentrations. The plates were incubated at 30°C. Colonies on
rich
plates appeared after 1 to 2 days, and those on Tol plates
were visible
after 3 to 4 days.
The different strains in the mixed fed-batch culture were distinguished
on NB plates by colony morphology and growth rate
and by plating
diluted culture samples on selective plates followed
by counting the
CFU on each plate. The selective plates were made
by adding a
particular volatile carbon source on a paper filter
disk which was
placed in the cover of MMY plates 1 day after plating.
The plates were
incubated at 30°C. Colonies appeared after 3 to
4 days. Of the four
strains studied, only
P. putida GJ31 is able
to grow on
chlorobenzene plates.
P. putida mt-2 could be identified
by
growth on
p-xylene plates, and
B. cepacia G4
could be identified
by growth on
o-cresol plates. The number
of
P. putida F1 cells
was determined by the difference
between the viability on
m-cresol
plates (on which both
P. putida F1 and
B. cepacia G4 grow) and
the
viability on
o-cresol plates (on which
P. putida
F1 does not
grow). Also,
P. putida F1 and
B. cepacia G4 could be distinguished
on
m-cresol plates
because the colonies of
B. cepacia G4 were
larger than the
colonies of
P. putida F1.
The percentage of cells of each strain that formed colonies on NB
plates and were also able to grow on toluene and on a selective
substrate was measured by replica plating colonies obtained from
NB
plates onto Tol plates and onto selective plates. Replica-plated
colonies on Tol plates were screened for the presence of catechol
2,3-dioxygenase (C23O) by spraying the plates with a 100 mM catechol
solution. Positive colonies of
P. putida mt-2 turn yellow
due
to conversion of catechol to 2-hydroxymuconic semialdehyde (HMS)
(
36). Mutants unable to grow on any of the selective
substrates
were further analyzed for the ability to grow on other
substrates
in batch cultures containing 1 or 5 mM substrate in MMV.
Plasmid extractions were done by using a modified method of Kado and
Liu (
18), as described by Duetz et al. (
7).
Southern
hybridizations and chemiluminescent detection of plasmid DNA
digested
with
EcoRI,
SalI, or
XhoI
were performed with a digoxigenin-labeled
probe containing the promoter
region of the
meta operon (Pm) and
xylXYZ' or
containing
xylTE' (GenBank accession number
M64747)
as
described by the manufacturer (Boehringer, Mannheim, Germany).
The
probes were obtained from the plasmids pGSH3537 (2.8-kb
SacI-
KpnI
fragment) and pAW31 (1.2-kb
SalI fragment) (
5a).
The decay rate of the strains in the fed-batch culture exposed to TCE
was calculated by using the formula
xt/x0 =
eadt, in which
xt is the viability on a selective plate at time
t and
x0 is the viability on a
selective plate at time zero, when
TCE addition to the fed-batch
culture started.
ad is the specific
decay rate
(
2).
Protein profiles of the strains were made by centrifuging 60 µl of an
overnight NB culture, resuspending the pellet in 10
µl of 1× loading
buffer (
31), and applying the mixture after
boiling (5 min)
on a 12.5% polyacrylamide gel containing sodium
dodecyl sulfate. The
gels were stained with Coomassie brilliant
blue.
Enzyme assays.
Mutants of P. putida mt-2 were
analyzed for the presence of enzymes of the meta- and
ortho-cleavage pathways. For this, cells were grown
overnight on 1 mmol of toluene h1 in a 2.5-liter
fed-batch culture. Cells were harvested by centrifugation and washed
with either ice-cold mineral medium (MM) or ice-cold 0.1 M Tris-HCl (pH
7.5) containing 0.1 mM 1,4-dithiothreitol (TD). After resuspension of
the cells in a small volume of MM or TD, they were used for the enzyme
assays, which were performed at 30°C.
The cells which were washed and resuspended in MM were used immediately
for oxygen uptake experiments. For this, cells were
added to a small,
magnetically stirred incubation vessel to which
an oxygen electrode
(O
2 sensor type 12/220; Ingold, Urdorf, Switzerland)
was
connected. After determination of the endogenous oxygen consumption
rate of the resting cell suspension, a concentrated solution of
benzoate,
m-toluate, or
p-toluate was added to
give a final concentration
of 5 mM. The difference between the oxygen
consumption rates before
and after addition of the substrate was used
to calculate the
specific rate of oxidation of the substrate in
micromoles gram
of cells (dry weight)
1
minute
1.
The cells which were washed and resuspended in TD were disrupted by
sonification and centrifuged for 30 min in an Eppendorf
centrifuge
(10,000 ×
g, 4°C). The clear supernatant solution
was
used as a source of crude cell extract. The protein content of
the
extract was determined with Coomassie brilliant blue, with
bovine serum
albumin as a standard.
Catechol 1,2-dioxygenase (C12O), C23O, 2-hydroxymuconic semialdehyde
hydrolase (HMSH), and 2-hydroxymuconic semialdehyde dehydrogenase
(HMSD) activities in the crude cell extract were measured as described
previously (
23). The conversion of catechol, 3MC, and
4-methylcatechol
(4MC) to their corresponding
meta-cleavage
products was monitored
at 375, 388, and 382 nm respectively. The
extinction coefficients
of HMS, 2-hydroxy-6-oxohepta-2,4-dienoic acid
(HODA), and 2-hydroxy-5-methylmuconic
semialdehyde (HMMS) are 36,000, 16,800, and 31,500 liters mol
1 cm
1,
respectively (
30). HMS, HODA, and HMMS were prepared in situ
by incubation of solutions containing catechol, 3MC, or 4MC,
respectively,
in 45 mM phosphate buffer (pH 7.4) with purified C23O of
P. putida mt-2 and were used to determine HMSH and HMSD
activities. C23O
was purified from a crude extract of
P. putida mt-2 by using an
acetone precipitation step followed by
ion-exchange and hydrophobic
interaction chromatographies.
Analytical methods.
TCE and toluene were measured in the gas
phase on a CP 9001 gas chromatograph equipped with a CP Sil 5 CB column
(length, 25 m; diameter, 0.53 mm) (Chrompack, Middelburg, The
Netherlands) and a flame ionization detector. The carrier gas (helium)
pressure was 150 kPa, and the oven temperature was 100°C.
Chloride was measured with a colorimetric assay (
3).
Estimation of kinetic parameters.
The kinetic parameters
(Ks and µmax) of the strains used
in the mixed fed-batch culture were estimated from substrate depletion curves obtained with growing batch cultures (29). For this, the strains were grown on toluene in a 3-liter bioreactor on 0.75 liter
of mineral medium at 30°C at a stirring speed of 1,050 rpm. The
depletion of toluene was measured by on-line analysis of the toluene
concentration in the headspace of the batch culture by gas
chromatography. The headspace was continuously circulated at a rate of
100 to 120 ml min1 with a micromembrane pump (NMP 02LU;
KNF Neuberger GmbH, Freiburg-Munzingen, Germany). After passage through
a Valco six-port sampling injector (Vici AG, Schenkon, Switzerland) to
which a 35-µl sample loop was connected, the gas was injected back
into the culture. Samples of 35 µl were automatically injected into
the gas chromatograph every 5 min.
The obtained substrate depletion curves were described with a model in
which the Monod equation and gas-liquid mass transfer
of the substrate
are incorporated, with biomass (
X) and gas and
liquid phase
concentrations (
Cg and
Cl) as variables. The volumes
of the gas and
liquid phases (
Vg and
Vl)
were 0.75 and 2.25 liters,
respectively. Values between 0.048 and 0.057 mg of cells (dry
weight) µmol
1 were taken for the yield
(
Y) of the different strains and were
determined from batch
cultures of each strain growing on 1 mM
toluene. The dimensionless
Henry's coefficient (
H) was determined
as described by Diks
(
6) (
Htoluene, 30°C = 0.27;
HTCE, 30°C = 0.5). The mass transfer
coefficient (
kLa) for toluene was determined
to
be 0.14 min
1 by a procedure described by van Hylckama
Vlieg et al. (
33).
The model consists of three equations:
X =
X0 + [(
Cg,0 
Cg)
Vg + (
Cl,0 Cl)
Vl]
Y/Vl,
dCg/dt =
kLa(
Cg/H Cl)
Vl/Vg,
and
dCl/dt =
kLa(
Cg/H Cl)
µ
maxCl/(
Cl +
Ks)
X/Y.
The parameters
Ks, µ
max, and the
initial concentrations of toluene in the gas and liquid phases
(
Cg,0 and
Cl,0)
were
fitted to the numerically integrated equations by using the
episode
routine in Scientist for Windows 2.0 (Micromath Scientific
Software,
Salt Lake City, Utah). The square of the difference
between the
measured and fitted values was multiplied by
1/(
Cg + 0.1) at each time point. The sum of
these relative squares was
minimized. This way, the data points at
lower toluene concentrations
have the same weight as the data points at
higher toluene concentrations,
while the more inaccurate values close
to the detection limit
(35 nM) are less important. The data points from
the first hour
were usually omitted to ensure that the system was in
equilibrium.
The TCE-degrading capacities of the strains were tested with
toluene-grown cells obtained from a 2-liter overnight fed-batch
culture
grown at a toluene addition rate of about 500 µmol h
1
to a density of 0.15 to 0.4 mg (dry weight) ml
1. Cells
were harvested by centrifugation and resuspended in MM
to a final
volume of 25 ml. The depletion of TCE was measured
by on-line analysis
of the TCE concentration in the headspace
of the resting
cell-suspension, which was placed in a 120-ml stainless
steel
incubation vessel at 30°C. TCE concentrations were determined
every
minute in the same way as for the toluene depletion experiments
described above. The culture was stirred at 1,000 rpm. The first-order
rate constant of TCE degradation, which is equal to the maximum
rate of
substrate conversion (
rmax) divided by the
Michaelis-Menten
constant (
Km), was determined
from the first-order region of the
TCE depletion curve. This was done
by plotting the natural logarithm
of the TCE concentration in the gas
phase against time and multiplying
the slope of this line by
(
VgH +
Vl)/
XVl.
| |
RESULTS |
Competition for toluene.
The competitive capacities of the
toluene-degrading strains P. putida GJ31, P. putida mt-2, P. putida F1, and B. cepacia G4 were studied with a mixed fed-batch culture at a very
low toluene concentration. For this, cells from batch cultures of each
strain were added to the fed-batch bioreactor, and the mixed culture was grown at a toluene addition rate of about 600 µmol
h1 until the culture reached an OD450 of 3. The toluene load was then reduced to 44 µmol h1.
Previous experiments with B. cepacia G4 had shown that at
this combination of toluene load and culture density, the specific growth rate on toluene is very low (22).
During 2 weeks, the culture density of the mixed fed-batch culture
slowly increased to an OD
450 of 4, after which it became
constant (Fig.
1), meaning that hardly
any net growth occurred
and that all toluene added was used for
maintenance of the culture.
After approximately 1 week of slow growth,
no toluene could be
detected in the outgoing gas stream (detection
limit, 35 nM),
which means that more than 99.7% of the added toluene
was converted
(Fig.
2A) and that growth
was not limited due to exhaustion of
other nutrients. The dry weight of
the culture was determined
after 40 days, and the toluene conversion of
the hardly growing,
mixed fed-batch culture was calculated to be 31 nmol mg of cells
(dry weight)
1 h
1.
View larger version (13K):
[in this window]
[in a new window]
|
FIG. 1.
Culture density of the fed-batch culture cultivated on
toluene in the absence and presence of TCE. The OD and protein
concentration were determined as a measure of the culture density.
Symbols:  , OD450;  , protein concentration;  , time
at which TCE addition was started.
|
|
View larger version (31K):
[in this window]
[in a new window]
|
FIG. 2.
Conversion of toluene (A) and TCE (B) by the fed-batch
culture. The concentrations of toluene and TCE in the ingoing and
outgoing gas streams were determined by gas chromatography. The
chloride concentration in the culture medium was determined with a
colorimetric assay. Symbols:  , toluene in the ingoing gas stream;
 , toluene in the outgoing gas stream;  , percentage of toluene
degraded by the culture; , TCE in the ingoing gas stream;  , TCE
in the outgoing gas stream;  , percentage of TCE degraded by the
culture (calculated from the concentrations of TCE in the in- and
outgoing gas streams);  , chloride concentration; , percentage of
TCE degraded by the fed-batch culture (calculated from chloride
measurements);  , time at which TCE addition was started.
|
|
The population composition of the culture was determined by counting
the colonies on selective plates (Fig.
3A). This corresponded
to the viability
of each strain on NB plates, which could be determined
based on colony
morphology. Just after the reduction of the toluene
load from about 600 to 44 µmol h
1, the contribution of each strain to the
total viability of the
mixed culture varied between 4% (for
P. putida mt-2) and 64% (for
P. putida F1). During the
cultivation at a very low toluene concentration,
the population
composition hardly changed, and all four strains
were maintained for at
least 40 days in the culture. The sum of
the viabilities of the strains
on selective plates was similar
to the total viability of the mixed
culture, as measured on NB
and Tol plates (Fig.
3B). This means that
all four strains were
able to grow on their selective substrate. This
was confirmed
by replica plating colonies derived from NB plates to
selective
plates.
View larger version (24K):
[in this window]
[in a new window]
|
FIG. 3.
Population composition (A) and viability (B) of the
fed-batch culture over time, determined as CFU on different agar
plates. Symbols: , P. putida GJ31 on chlorobenzene
plates; , P. putida mt-2 on p-xylene plates;
, B. cepacia G4 on o-cresol plates; ,
P. putida F1, determined as the difference between the
viability on m-cresol and o-cresol plates; ,
viability on NB plates,  , viability on Tol plates; , sum of
viabilities of the four strains on selective plates; , time at which
TCE addition was started.
|
|
Competition for toluene in the presence of TCE.
To study the
influence of TCE on the competitive behavior of the four strains at
very low toluene concentrations, TCE was added to the culture at a rate
of 24 µmol h1, starting after 40 days of fed-batch
cultivation on toluene. During TCE addition, the culture density slowly
decreased to an OD450 of 3.6 at 83 days. Immediately after
the start of TCE addition, the toluene concentration in the outgoing
gas stream increased to a maximum of around 500 nM after 2 days of TCE
addition. It then slowly decreased to approximately twice the detection
limit (Fig. 2A). Approximately 17% of the incoming TCE was degraded after 1 day, but this became less than 3% after 4 days (Fig. 2B). The
chloride concentration slowly increased in the fed-batch culture (Fig.
2B).
Within a few days after the start of the addition of TCE, the viability
of
B. cepacia G4, measured as CFU on
o-cresol
plates,
decreased with a specific decay rate of 0.0037 h
1
(
r2 = 0.70).
P. putida GJ31 and
P. putida F1 showed similar behavior.
The viability on
chlorobenzene plates and the difference in viability
on
m-cresol plates and
o-cresol plates decreased
with specific
decay rates of 0.0064 (
r2 = 0.84)
and 0.0068 (
r2 = 0.94) h
1,
respectively. The viability of
P. putida mt-2 on
p-xylene plates
remained at a constant level (Fig.
3A).
These results show that
only a small amount of TCE could be converted
by the mixed culture
and that the TCE degradation capacity decreased
over time, since
the TCE-converting organisms were outcompeted by
P. putida mt-2.
Accumulation of mutants.
After 1 week of TCE addition, a
difference arose between the sum of the viabilities of the strains on
selective plates and the viabilities of the mixed culture on NB plates
and on Tol plates (Fig. 3B). To check if all the cells were still able
to grow on one of the selective substrates, colonies derived from NB
plates were replica plated on selective plates. An increasing amount of
colonies was no longer able to grow on p-xylene,
o-cresol, m-cresol, or chlorobenzene, indicating
that these colonies are mutants.
The colony morphologies of these mutants were similar to that of
P. putida mt-2. The protein profiles of the mutants were
also found to be similar to the profile of the wild-type
P. putida mt-2 by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis
(data not shown), indicating that they were mutants of
P. putida mt-2. The mutants were further examined by replica
plating on
Tol plates and on the selective plates. Cells grown on Tol
plates
were checked for C23O (XylE) activity by spraying with catechol.
If XylE is active, the colonies rapidly become yellow due to the
conversion of catechol to the yellow HMS.
Three classes of mutants were distinguished. One class of mutants was
no longer able to grow on toluene or on any of the other
selective
substrates. The mutants possessed a plasmid that was
much smaller than
the wild-type TOL plasmid. It did not hybridize
with the
Pm-
xylXYZ' and
xylTE' probes, indicating that it
lacks
the 39-kb fragment encoding the catabolic genes for toluene and
xylene growth. Loss of this fragment also occurs during growth
on
benzoate (see, e.g., reference
37). Immediately
after the
addition of TCE, this phenotype was observed for
approximately
20% of the
P. putida mt-2-like colonies.
However, this class of
mutants disappeared after 1 week (Fig.
4).
View larger version (18K):
[in this window]
[in a new window]
|
FIG. 4.
Appearance of mutants of P. putida mt-2
during fed-batch cultivation. Colonies with a morphology similar to
that of P. putida mt-2 on NB plates were replica plated on
Tol plates and selective plates to determine their phenotype. Activity
of XylE was determined by spraying Tol plates with replica-plated
colonies with a catechol solution and checking for the formation of the
yellow product HMS. Symbols: , colonies able to grow on
p-xylene and toluene and forming HMS after being sprayed
with catechol (Tol+ p-Xyl+
XylE+ [wild-type]); , colonies unable to grow on
p-xylene and toluene (Tol
p-Xyl); , colonies able to grow on toluene,
unable to grow on p-xylene, and not forming HMS when sprayed
with catechol (Tol+ p-Xyl
XylE); , colonies able to grow on toluene, unable to
grow on p-xylene, and forming of HMS after being sprayed
with catechol (Tol+ p-Xyl
XylE+); , time at which TCE addition was started.
|
|
A second class of mutants was XylE
+ and could still grow on
toluene,
m-xylene, and
m-toluate but not on
p-xylene or
p-toluate.
Approximately one-third of
the mutants which could still grow
on toluene but not on the selective
plates belonged to this class
(Tol
+
p-Xyl
XylE
+) (Fig.
4). The
specific rate at which these mutants appeared
was estimated from the
increase in their number over time by multiplying
the number of
P. putida mt-2-like colonies on NB plates by the
percentage of Tol
+ p-Xyl
XylE
+ mutants, which was determined by replica plating at
each time
point, and was found to be 0.032 h
1
(
r2 = 0.65). No differences in plasmid size
could be seen on agarose
gels, and the plasmid still hybridized with
the
xylTE' and Pm-
xylXYZ'
probes. Southern
hybridizations of
EcoRI-,
XhoI-, or
SalI-digested
plasmid DNA with the Pm-
xylXYZ' and
xylTE' probes also revealed
no differences. However, in a
crude extract of toluene-grown cells
of one of these mutants (
P. putida mt-2M11), the activities of
three enzymes of the
meta-cleavage pathway with different substrates
were
approximately one-fifth of the activities found with wild-type
cells
(Table
2). Also, cells of
P. putida mt-2M11 which were
grown on toluene no longer oxidized
p-toluate (Table
3). In
P. putida mt-2,
p-toluate is oxidized by a
toluate dioxygenase (XylXYZ)
which can also convert benzoate and
m-toluate. The
xylXYZ genes
are located in the
meta operon of the TOL plasmid of
P. putida mt-2,
directly behind the promoter of the operon (
1). The
chromosomally
encoded equivalent of XylXYZ is a benzoate dioxygenase,
which
can convert only benzoate and
m-toluate (
15,
28). The data
suggest that the mutants of class 2 have a small
mutation in the
promoter region of the
meta operon, which
could not be detected
by Southern analysis but which influences the
expression of the
meta pathway and leads to the absence of
activity of XylXYZ.
View this table:
[in this window]
[in a new window]
|
TABLE 2.
Specific activities of enzymes of the ortho
and meta pathways in crude extracts of P. putida
mt-2 and mutants that originated from this strain
|
|
View this table:
[in this window]
[in a new window]
|
TABLE 3.
Oxygen uptake by washed resting cell suspensions of
P. putida mt-2 and of mutants which originated from
this strain
|
|
The third class of mutants was still able to grow on toluene but not on
p-xylene or
p-toluate. When colonies of this
class
were sprayed with catechol, little or no yellow coloration
occurred.
The specific rate at which these Tol
+
p-Xyl
XylE
mutants appeared was
0.064 h
1 (
r2 = 0.90). Most of
these mutants grew very poorly on
m-xylene and
m-toluate, while the medium became brown, which indicates
the
accumulation of a (substituted) catechol. One of the mutants
(
P. putida mt-2M4) was unable to grow on
m-xylene
at all. It had a
plasmid which was slightly smaller than the TOL
plasmid of
P. putida mt-2 and did not hybridize with a
Pm-
xylXYZ' or a
xylTE'
probe. No activity of
enzymes of the
meta pathway could be detected
(Table
2).
This suggested that
P. putida mt-2M4 lacks all or
a large
part of the genes of the
meta-cleavage pathway. A similar
mutation was found by Brinkmann et al. (
4) during unlimited
growth of
P. putida on toluene.
The other mutants of the third phenotypic class contained a plasmid
which still hybridized with both probes and seemed to
have the same
size as the wild-type plasmid. However, Southern
analysis showed that
both the
SalI and
EcoRI fragments of the
plasmid
encoding part of
xylX and the promoter region were about
0.4 kb smaller than the corresponding wild-type fragments. The
activities
of enzymes of the
meta pathway were largely reduced
in a
crude extract of a toluene-grown mutant of this class (
P. putida mt-2M10) (Table
2), indicating that the deletion caused
a
large reduction of the expression of the
meta pathway. None
of the mutants of class 3 could oxidize
p-toluate (Table
3),
indicating that XylXYZ is not active in these cells.
Kinetic analysis of toluene-degrading strains.
To determine
the kinetic basis for the observed population changes and appearance of
mutants, the kinetic parameters (µmax and
Ks) of each strain were determined by on-line
gas chromatographic analysis of toluene depletion from the headspaces
of batch cultures. An example of a depletion curve is presented in Fig.
5A. The data show that B. cepacia G4 has a much lower affinity
(µmax/Ks) for toluene than
the other three strains, while P. putida GJ31 has the best kinetics for growth on toluene (Table
4).
View larger version (27K):
[in this window]
[in a new window]
|
FIG. 5.
Toluene depletion curves obtained with batch cultures of
P. putida mt-2 growing on toluene in the absence of TCE (A)
and P. putida GJ31 growing on toluene in the presence of TCE
(B). The concentrations of toluene () and TCE () in the headspace
of the culture were determined. The lines through the open circles are
the fitted toluene depletion curves. The other lines show the deviation
of the data points from the fitted curve.
|
|
The TCE-degrading capacities of the strains were determined from TCE
depletion curves measured with toluene-grown cells (Fig.
6). The first-order rate constants of TCE
degradation were 52
and 0.6 ml mg of cells (dry weight)
1
h
1 for
B. cepacia G4 and
P. putida
GJ31, respectively. For
P. putida F1 the rate
constant of TCE degradation decreased rapidly from
14 to 1.7 ml mg of
cells (dry weight)
1 h
1. Wackett and
coworkers also observed a rapid decrease of the
rate over time, which
was probably caused by the formation of
toxic intermediates (
34,
35).
P. putida mt-2 showed no detectable
TCE
degradation (Fig.
6). The data show that
P. putida mt-2 is
not able to degrade TCE, while
P. putida GJ31 degrades TCE
much
slower than
P. putida F1 and
B. cepacia G4,
which are well known
for their TCE-degrading capacities.
View larger version (20K):
[in this window]
[in a new window]
|
FIG. 6.
Logarithmic plots of TCE depletion by resting cell
suspensions of B. cepacia G4 and P. putida F1,
GJ31, and mt-2. The cells were grown on toluene. The concentrations of
TCE in the headspace of the cultures were measured. TCE depletion by
0.43 mg (dry weight) of B. cepacia G4 ml1
(), 1.0 mg (dry weight) of P. putida F1 ml1
(), 1.9 mg (dry weight) of P. putida GJ31
ml1 (), and 3.9 mg (dry weight) of P. putida mt-2 ml1 () is shown. The lines show the
TCE depletion curves that were fitted through the data located in
linear regions of the curves for B. cepacia G4, P. putida F1, and P. putida GJ31.
|
|
To study the effect of TCE on the kinetics of toluene utilization,
toluene depletion curves were made in the presence of TCE,
which was
added to similar concentrations as with the fed-batch
culture.
B. cepacia G4 significantly converted TCE during growth
on toluene,
whereas the other three strains hardly converted TCE
(Fig.
5B). The
apparent affinities for toluene decreased in the
presence of TCE (Table
4). For
P. putida mt-2, this decrease
was twofold. However,
the affinities for toluene of
P. putida F1 and GJ31 became
almost 1 order of magnitude lower in the presence
of TCE. Together with
B. cepacia G4, these strains disappeared
from the fed-batch
culture after the start of TCE addition and
are able to (slightly)
degrade TCE.
The TCE depletion curve of
P. putida GJ31 showed first-order
behavior. This means that the half-saturation constant
(
KmTCE) of TCE conversion is much
higher than the initial TCE concentration
in the experiment, which was
18 µM in the liquid phase. TCE was
present at 11 µM in the liquid
phase during the preparation of
the toluene depletion curve for strain
GJ31. With these numbers,
a model for competitive inhibition
{
Ksobs =
Ks(1 + [TCE]/
Ki)} predicts an increase in the
observed Monod
constants (
Ksobs)
with a factor of less than 1.6 if
Ki were
identical to
KmTCE. However, the
increase in
Ksobs which was
determined from toluene depletion curves in the presence
of TCE was
much higher (Table
4), indicating that the inhibition
of toluene
utilization by TCE cannot be described by a simple
model for
competitive inhibition. Landa et al. (
19) also found
that
this model could not describe cometabolic TCE degradation
by
B. cepacia G4 in continuous culture. Instead, they observed
that the
inhibition constant of TCE for the conversion of toluene
was higher
than the
Km of TCE conversion.
The kinetic parameters for growth on toluene of some mutants of
P. putida mt-2 are given in Table
5. Mutants belonging to
both class 2 and
class 3 were analyzed. All five mutants had a
higher µ
max
and a higher affinity than wild-type
P. putida mt-2.
The presence of TCE still caused a small decrease in the affinity
for
toluene (data not shown).
| |
DISCUSSION |
The four toluene-degrading strains used in this study
(B. cepacia G4, P. putida mt-2, P. putida GJ31, and P. putida F1) had to compete for very
low concentrations of toluene when they were cultivated together in a
fed-batch culture. In situations like this, hardly any growth of the
culture is allowed. However, shifts in the population composition are
still possible. Zambrano et al. (38), for example, described
the takeover of a stationary culture by a mutant of Escherichia
coli. Likewise, we observed that mutants which had lost pTOM took
over a culture of B. cepacia G4 which was exposed to TCE
while being starved for carbon and energy (22). Under the
conditions that were used here, all four strains were able to maintain
themselves in the fed-batch culture at a rather constant viability for
at least 40 days.
The strains that were present in the fed-batch culture showed
large differences in the individual kinetic parameters
µmax and Ks. If the capacities of
the strains to compete during severe toluene limitation were determined
by their kinetic properties, one would expect the strain with the
highest affinity for toluene to take over the culture. Since no
important changes in the population composition were observed, these
kinetic parameters did not predict the outcome of the competition. This
means that other factors also determined the competitive capacities of
the strains. Bacteria could, for example, increase their affinity
for toluene by a stronger induction of the enzymes involved in toluene
degradation (20) or could produce compounds which inhibit
other species. Factors like the amount of energy needed for
maintenance purposes and the growth yield on products of
lysed cells can also influence the survival of the strains. Also
in continuous culture, the outcome of competition
experiments cannot always be predicted by the affinity of each
strain. Despite the lower affinity of B. cepacia
G4 for toluene, it could win over P. putida mt-2
(8).
After TCE addition, only P. putida mt-2, which does not
degrade TCE, remained in the culture. The numbers of cells of B. cepacia G4, P. putida F1, and P. putida GJ31
decreased, and TCE degradation diminished rapidly. We previously found
that a nongrowing culture of B. cepacia G4 could
cometabolically degrade TCE in a fed-batch culture as long as toluene
was added (22). However, this resulted in a large increase
in the maintenance energy demand of B. cepacia G4, most
likely due to toxic effects of TCE conversion. Cytotoxicity by TCE
conversion products has also been observed for P. putida F1
(35). Such toxic effects could give TCE-converting organisms a selective disadvantage, especially at the low toluene concentrations used.
Compared to that of B. cepacia G4, the first-order rate
constant for TCE degradation of P. putida GJ31 suggests that
at least 85 times less TCE will be converted by this strain, which is
likely to be even less because of its superior kinetics for toluene
degradation. In spite of that, the observed decay rates for P. putida GJ31 and F1 after TCE addition were even higher than the
decay rate for B. cepacia G4. Analysis of toluene depletion
curves made in the presence of concentrations of TCE similar to that in
the fed-batch culture showed that the affinity
(µmax/Ksobs) of
P. putida GJ31 for toluene strongly decreased. Such a
decrease was also observed for P. putida F1. For
B. cepacia G4 the affinity decreased only 2.2-fold.
However, during the kinetic measurements, B. cepacia G4
degraded a considerable amount of TCE, which leads to a large
underestimation of Ksobs.
Although the affinity for toluene is not the sole parameter determining
the outcome of the competition, reduction of affinity could still lower
the competitiveness of an individual strain. Quantitatively, the effect
of TCE on the Ksobs for toluene as
determined in depletion experiments could not be described by
competitive inhibition. For P. putida GJ31 and F1, the
effect of TCE was larger than expected, which may be due to enzyme
inactivation or product inhibition in addition to competitive inhibition. Our results indicate that the actual amount of TCE that can
be converted by the organisms is not the only factor that determines
their fate in the population, because the kinetics for the degradation
of TCE did not correspond to the effect of this compound on the
kinetics for growth on toluene and on the survival of the different
strains.
After TCE addition was started, the competitive capacity of P. putida mt-2 was further improved by mutations which allowed this
organism to grow on toluene at a higher rate, but this also resulted in
the loss of the capacity to grow on p-xylene. The mutants
did not convert p-toluate, indicating that they do not have
any active XylXYZ. Also, the expression levels of enzymes further down
in the meta pathway were strongly reduced. When the mutants
grow on toluene, the enzymes of the upper pathway (1) will
convert toluene to benzoate, after which the absence of XylXYZ and the
reduction of the expression of the meta pathway probably allow benzoate to be degraded mainly via the ortho pathway,
which is known to result in a higher growth rate (1, 4). The
p-Xyl mutants were detected after 2 days of
TCE addition (Fig. 4). By that time, the toluene concentration in the
outgoing gas stream had increased to ~500 nM, probably due to the
decay of B. cepacia G4 and P. putida F1 and GJ31.
Since the mutants of P. putida mt-2 have an elevated growth
rate on toluene, they could probably take over the culture more rapidly
than wild-type P. putida mt-2.
During the 40 days of toluene addition in the absence of TCE, no
p-Xyl mutants were detected, although they
could have had an improved fitness compared to P. putida
mt-2. Since the mutants were observed soon after the start of TCE
addition, the appearance of the mutants seemed to be a direct effect of
this, for example, because TCE has some mutagenic effect which
increases the overall rate of mutations or because TCE specifically
inhibits a component of wild-type P. putida mt-2. XylXYZ
might be such a component, since none of the mutants of the different
classes had activity of this dioxygenase, while the expression levels
of other enzymes of the meta operon differed considerably.
Although several studies describe the potential of toluene and phenol
degraders for successful remediation of TCE (10, 12-14), our results indicate that the application of microorganisms that cometabolically degrade TCE carries a high risk of takeover of the
desired population by organisms that are less sensitive to inhibitory
effects of TCE. Fries et al. (11) showed that there is a
large variety in the capacity to degrade TCE among toluene- and
phenol-degrading microorganisms isolated from the Moffett field, and
they also expected that organisms which do not degrade TCE will
eventually take over the population. Indeed, Munakata-Marr et al.
(24) recently observed a gradual decline in the breakdown of
TCE in phenol-fed microcosms containing aquifer material from the
Moffett field, while degradation of phenol remained complete. This was
probably caused by a shift in the population towards phenol degraders
that did not degrade TCE.
Stable degradation may require the stimulation of a specific group of
organisms. This might be achieved by using a less common primary
substrate which can be degraded only by enzymes that also convert TCE
and for which no alternatives exist. o-Cresol might be such
a primary substrate. It is degraded by the same TCE-degrading toluene
monooxygenase (TOM) as toluene in B. cepacia G4
(32). TOM-containing organisms were found to dominate in the
TCE-contaminated Moffett field (11), which indicates that
the endogenous population can degrade TCE with o-cresol. In
case of groundwater treatment with continuously operated bioreactors,
separation of degradation and growth is another alternative to overcome
instability problems and is currently under study in our lab.
| |
ACKNOWLEDGMENTS |
This work was financed by grants from the Dutch IOP Environmental
Biotechnology program and the EC environment program.
We acknowledge Uwe Dehmel for providing the plasmids with the
xylTE' and Pm-xylXYZ' genes. Wouter Duetz
assisted with the P. putida mt-2 plasmid
isolations.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG
Groningen, The Netherlands. Phone: 31-503634008. Fax: 31-503634165. E-mail: D.B.Janssen{at}chem.rug.nl.
| |
REFERENCES |
| 1.
|
Assinder, S. J., and P. A. Williams.
1990.
The TOL plasmids: determinants of the catabolism of toluene and the xylenes.
Adv. Microb. Physiol.
31:1-69[Medline].
|
| 2.
|
Bailey, J. E., and D. F. Ollis.
1986.
.
Biochemical engineering fundamentals, 2nd ed.
McGraw-Hill Inc., New York, N.Y.
|
| 3.
|
Bergmann, J. G., and J. Sanik.
1957.
Determination of trace amounts of chlorine in naphtha.
Anal. Chem.
29:241-243.
|
| 4.
|
Brinkmann, U.,
J. L. Ramos, and W. Reineke.
1994.
Loss of the TOL meta-cleavage pathway functions of Pseudomonas putida strain PaW1 (pWWO) during growth on toluene.
J. Basic Microbiol.
5:303-309.
|
| 5.
|
Chesbro, W.
1988.
The domains of slow bacterial growth.
Can. J. Microbiol.
34:427-435[Medline].
|
| 5a.
| Dehmel, U. Personal communication.
|
| 6.
|
Diks, R. M. M.
1992.
.
The removal of dichloromethane from waste gases in a biological trickling filter. Ph.D. thesis.
Eindhoven University of Technology, Eindhoven, The Netherlands.
|
| 7.
|
Duetz, W. A.,
M. K. Winson,
J. G. van Andel, and P. A. Williams.
1991.
Mathematical analysis of catabolic function loss in a population of Pseudomonas putida mt-2 during non-limited growth on benzoate.
J. Gen. Microbiol.
137:1363-1368[Abstract/Free Full Text].
|
| 8.
|
Duetz, W. A.,
C. de Jong,
P. A. Williams, and J. G. van Andel.
1994.
Competition in chemostat culture between Pseudomonas strains that use different pathways for the degradation of toluene.
Appl. Environ. Microbiol.
60:2858-2863[Abstract/Free Full Text].
|
| 9.
|
Ensley, B. D.
1991.
Biochemical diversity of trichloroethylene metabolism.
Annu. Rev. Microbiol.
45:283-299[Medline].
|
| 10.
|
Fan, S., and K. M. Scow.
1993.
Biodegradation of trichloroethylene and toluene by indigenous microbial populations in soil.
Appl. Environ. Microbiol.
59:1911-1918[Abstract/Free Full Text].
|
| 11.
|
Fries, M. R.,
L. J. Forney, and J. M. Tiedje.
1997.
Phenol- and toluene-degrading microbial populations from an aquifer in which successful trichloroethene cometabolism occurred.
Appl. Environ. Microbiol.
63:1523-1530[Abstract].
|
| 12.
|
Hopkins, G. D.,
J. Munakata,
L. Semprini, and P. L. McCarty.
1993.
Trichloroethylene concentration effects on pilot-scale in-situ groundwater bioremediation by phenol-oxidizing microorganisms.
Environ. Sci. Technol.
27:2542-2547.
|
| 13.
|
Hopkins, G. D.,
L. Semprini, and P. L. McCarty.
1993.
Microcosm and in situ field studies of enhanced biotransformations of trichloroethylene by phenol-utilizing microorganisms.
Appl. Environ. Microbiol.
59:2277-2285[Abstract/Free Full Text].
|
| 14.
|
Hopkins, G. D., and P. L. McCarty.
1995.
Field evaluation of in situ aerobic cometabolism of trichloroethylene and three dichloroethylene isomers using phenol and toluene as the primary substrates.
Environ. Sci. Technol.
29:1628-1637.
|
| 15.
|
Ichihara, A.,
K. Adachi,
K. Hosokawa, and Y. Takeda.
1962.
The enzymatic hydroxylation of aromatic carboxylic acids; substrate specificities of anthranilate and benzoate oxidases.
J. Biol. Chem.
237:2297-2302.
|
| 16.
|
Janssen, D. B.,
A. Scheper, and B. Witholt.
1984.
Biodegradation of 2-chloroethanol by pure bacterial cultures.
Prog. Ind. Microbiol.
20:169-178.
|
| 17.
|
Janssen, D. B., and B. Witholt.
1992.
Aerobic and anaerobic degradation of halogenated aliphatics, p. 299-327. In
H. Sigel, and A. Sigel (ed.), Metal ions in biological systems, vol. 28. Degradation of environmental pollutants by microorganisms and their metalloenzymes.
Marcel Dekker, Inc., New York, N.Y.
|
| 18.
|
Kado, C. I., and S.-T. Liu.
1981.
Rapid procedure for detection and isolation of large and small plasmids.
J. Bacteriol.
145:1365-1373[Abstract/Free Full Text].
|
| 19.
|
Landa, A. S.,
E. M. Sipkema,
J. Weijma,
A. A. C. M. Beenackers,
J. Dolfing, and D. B. Janssen.
1994.
Cometabolic degradation of trichloroethylene by Pseudomonas cepacia G4 in a chemostat with toluene as the primary substrate.
Appl. Environ. Microbiol.
60:3368-3374[Abstract/Free Full Text].
|
| 20.
|
Law, A. T., and D. K. Button.
1986.
Modulation of affinity of a marine pseudomonad for toluene and benzene by hydrocarbon exposure.
Appl. Environ. Microbiol.
51:469-476[Abstract/Free Full Text].
|
| 21.
|
Lowry, O. H.,
N. J. Rosebrough,
A. L. Farr, and R. J. Randall.
1951.
Protein measurement with the Folin phenol reagent.
J. Biol. Chem.
193:265-275[Free Full Text].
|
| 22.
|
Mars, A. E.,
J. Houwing,
J. Dolfing, and D. B. Janssen.
1996.
Degradation of toluene and trichloroethylene by Burkholderia cepacia G4 in growth-limited fed-batch culture.
Appl. Environ. Microbiol.
62:886-891[Abstract].
|
| 23.
|
Mars, A. E.,
T. Kasberg,
S. R. Kaschabek,
M. H. van Agteren,
D. B. Janssen, and W. Reineke.
1997.
Microbial degradation of chloroaromatics: use of the meta-cleavage pathway for mineralization of chlorobenzene.
J. Bacteriol.
179:4530-4537[Abstract/Free Full Text].
|
| 24.
|
Munakata-Marr, J.,
V. G. Matheson,
L. J. Forney,
J. M. Tiedje, and P. L. McCarty.
1997.
Long-term biodegradation of trichloroethylene influenced by bioaugmentation and dissolved oxygen in aquifer microcosms.
Environ. Sci. Technol.
31:786-791.
|
| 25.
|
Nelson, M. J. K.,
S. O. Montgomery,
W. R. Mahaffey, and P. H. Pritchard.
1987.
Biodegradation of trichloroethylene and involvement of an aromatic pathway.
Appl. Environ. Microbiol.
53:949-954[Abstract/Free Full Text].
|
| 26.
|
Nelson, M. J. K.,
S. O. Montgomery,
O. J. O'Neill, and P. H. Pritchard.
1986.
Aerobic metabolism of trichloroethylene by a bacterial isolate.
Appl. Environ. Microbiol.
52:383-384[Abstract/Free Full Text].
|
| 27.
|
Oldenhuis, R.,
J. Y. Oedzes,
J. J. van der Waarde, and D. B. Janssen.
1991.
Kinetics of chlorinated hydrocarbon degradation by Methylosinus trichosporium OB3b and toxicity of trichloroethylene.
Appl. Environ. Microbiol.
57:7-14[Abstract/Free Full Text].
|
| 28.
|
Reineke, W., and H.-J. Knackmuss.
1978.
Chemical structure and biodegradability of halogenated aromatic compounds. Substituent effects on 1,2-dioxygenation of benzoic acid.
Biochim. Biophys. Acta
542:412-423[Medline].
|
| 29.
|
Robinson, J. A., and J. M. Tiedje.
1983.
Nonlinear estimation of Monod growth kinetic parameters from a single substrate depletion curve.
Appl. Environ. Microbiol.
45:1453-1458[Abstract/Free Full Text].
|
| 30.
|
Sala-Trepat, J. M.,
K. Murray, and P. A. Williams.
1972.
The metabolic divergence in the meta cleavage of catechols by Pseudomonas putida NCIB 10015. Physiological significance and evolutionary implications.
Eur. J. Biochem.
28:347-356[Medline].
|
| 31.
|
Sambrook, J.,
E. F. Fritsch, and T. Maniatis.
1989.
.
Molecular cloning: a laboratory manual.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 32.
|
Shields, M. S.,
S. O. Montgomery,
S. M. Cuskey,
P. J. Chapman, and P. H. Pritchard.
1991.
Mutants of Pseudomonas cepacia G4 defective in catabolism of aromatic compounds and trichloroethylene.
Appl. Environ. Microbiol.
57:1935-1941[Abstract/Free Full Text].
|
| 33.
|
van Hylckama Vlieg, J. E. T.,
W. de Koning, and D. B. Janssen.
1996.
Transformation kinetics of chlorinated ethenes by Methylosinus trichosporium OB3b and detection of unstable epoxides by on-line gas chromatography.
Appl. Environ. Microbiol.
62:3304-3312[Abstract].
|
| 34.
|
Wackett, L. P., and D. T. Gibson.
1988.
Degradation of trichloroethylene by toluene dioxygenase in whole-cell studies with Pseudomonas putida F1.
Appl. Environ. Microbiol.
54:1703-1708[Abstract/Free Full Text].
|
| 35.
|
Wackett, L. P., and S. R. Householder.
1989.
Toxicity of trichloroethylene to Pseudomonas putida F1 is mediated by toluene dioxygenase.
Appl. Environ. Microbiol.
55:2723-2725[Abstract/Free Full Text].
|
| 36.
|
Wigmore, G. J.,
R. C. Bayly, and D. Di Berardino.
1974.
Pseudomonas putida mutants defective in the metabolism of the products of meta fission of catechol and its methyl analogues.
J. Bacteriol.
120:416-423[Abstract/Free Full Text].
|
| 37.
|
Williams, P. A.,
S. Taylor, and L. E. Gibb.
1988.
Loss of the toluene-xylene catabolic genes of TOL plasmid pWWO during growth of Pseudomonas putida on benzoate is due to a selective growth advantage of `cured' segregants.
J. Gen. Microbiol.
134:2039-2048[Abstract/Free Full Text].
|
| 38.
|
Zambrano, M. M.,
D. A. Siegele,
M. Almiron,
A. Tormo, and R. Kolter.
1993.
Microbial competition: Escherichia coli mutants that take over stationary phase cultures.
Science
259:1757-1759[Abstract/Free Full Text].
|
Appl Environ Microbiol, January 1998, p. 208-215, Vol. 64, No. 1
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Futamata, H., Nagano, Y., Watanabe, K., Hiraishi, A.
(2005). Unique Kinetic Properties of Phenol-Degrading Variovorax Strains Responsible for Efficient Trichloroethylene Degradation in a Chemostat Enrichment Culture. Appl. Environ. Microbiol.
71: 904-911
[Abstract]
[Full Text]
-
Dinkla, I. J. T., Gabor, E. M., Janssen, D. B.
(2001). Effects of Iron Limitation on the Degradation of Toluene by Pseudomonas Strains Carrying the TOL (pWWO) Plasmid. Appl. Environ. Microbiol.
67: 3406-3412
[Abstract]
[Full Text]
-
Yeager, C. M., Bottomley, P. J., Arp, D. J.
(2001). Cytotoxicity Associated with Trichloroethylene Oxidation in Burkholderia cepacia G4. Appl. Environ. Microbiol.
67: 2107-2115
[Abstract]
[Full Text]
-
Mars, A. E., Kingma, J., Kaschabek, S. R., Reineke, W., Janssen, D. B.
(1999). Conversion of 3-Chlorocatechol by Various Catechol 2,3-Dioxygenases and Sequence Analysis of the Chlorocatechol Dioxygenase Region of Pseudomonas putida GJ31. J. Bacteriol.
181: 1309-1318
[Abstract]
[Full Text]
Top
Abstract
Introduction
