A method for detection of aromatic metabolites at very low concentrations: Application to detection of metabolites of anaerobic toluene degradation

Abstract

Difficulties inherent in working with anaerobic microorganisms and mixed cultures have hampered efforts to detect and identify metabolites of anaerobic degradation of monoaromatic compounds. Isotope-trapping experiments and analysis using a high-performance liquid chromatograph equipped with a flow-through radioactivity detector were used to detect very low concentrations of metabolites. Data obtained by this method suggest that toluene was degraded via methyl hydroxylation by a mixed methanogenic culture.

Full text

APPLIED
AND
ENVIRONMENTAL
MICROBIOLOGY,
Jan.
1994,
p.
323-327
0099-2240/94/$04.00+0
Copyright
X
1994,
American
Society
for
Microbiology
NOTES
A
Method
for
Detection
of
Aromatic
Metabolites
at
Very
Low
Concentrations:
Application
to
Detection
of
Metabolites
of
Anaerobic
Toluene
Degradationt
ELIZABETH
A.
EDWARDS,"*
ALED
M.
EDWARDS,2
AND
DUNJA
GRBIC&GALIC'
Environmental
Engineering
and
Science,
Department
of
Civil
Engineering,
Stanford
University,
Stanford,
California
94305-4020,1
and
Department
of
Patholog,
McMaster
University,
Hamilton,
Ontario
L8N
2Z5,
Canada
Received
19
May
1993/Accepted
2
November
1993
Difficulties
inherent
in
working
with
anaerobic
microorganisms
and
mixed
cultures
have
hampered
efforts
to
detect
and
identify
metabolites
of
anaerobic
degradation
of
monoaromatic
compounds.
Isotope-trapping
experiments
and
analysis
using
a
high-performance
liquid
chromatograph
equipped
with
a
flow-through
radioactivity
detector
were
used
to
detect
very
low
concentrations
of
metabolites.
Data
obtained
by
this
method
suggest
that
toluene
was
degraded
via
methyl
hydroxylation
by
a
mixed
methanogenic
culture.
Research
on
the
anaerobic
biodegradation
of
monoaro-
matic
hydrocarbons,
like
toluene,
has
been
hampered
by
difficulties
associated
with
studying
anaerobic
microorgan-
isms
(low
growth
rates,
poorly
defined
growth
requirements,
and
the
need
for
specialized
equipment).
The
detailed
bio-
chemical
analysis
of
anaerobic
biodegradation
is
plagued
by
additional
problems.
Strict
anaerobes
found
in
sulfate-reduc-
ing
and
methanogenic
cultures
are
inhibited
by
high
sub-
strate
concentrations
(for
toluene,
typically
less
than
400
,M),
and
thus
only
low
amounts
of
substrate
can
be
used
to
sustain
growth.
As
a
result
of
these
low
substrate
concen-
trations,
together
with
the
low
rates
of
growth
and
degrada-
tion,
and
possibly
the
relatively
small
amount
of
energy
available
from
the
reaction
(4, 5),
intermediates
in
the
catabolic
pathways
do
not
appear
to
accumulate
and
have
proven
very
difficult
to
detect.
A
high-performance
liquid
chromatography
(HPLC)-radioactive
tracer
method
pre-
sented
in
this
paper
offers
a
sensitive
way
to
detect
metab-
olites
at
very
low
concentrations.
Radioactive
tracing
and
isotope
trapping
are
very
effective
techniques
for
determin-
ing
metabolic
pathways,
especially
when
the
concentrations
are
very
low,
because
these
low
concentrations
can
be
overcome
by
using
a
radioactive
substrate
with
high
specific
activity.
Labeled
substrates
also
provide
an
indisputable
link
between
the
substrate
and
any
labeled
products
detected.
Our
method
was
applied
to
the
search
for
metabolites
of
anaerobic
toluene
degradation
by
a
mixed
methogenic
cul-
ture
(4).
The
data
which
we
present
suggest
that
toluene
degradation
by
this
methanogenic
culture
proceeded
via
methyl
hydroxylation
to
benzyl
alcohol,
followed
by
further
oxidation
steps
to
benzaldehyde
and
benzoate,
with
perhaps
a
parallel
pathway
via
ring
hydroxylation
to
p-cresol.
We
first
attempted
to
determine
intermediates
of
metha-
*
Corresponding
author.
Present
address:
Beak
Consultants
Ltd.,
42
Arrow
Rd.,
Guelph,
Ontario,
Canada,
N1K-1S6.
Phone:
519-763-
2325
x236.
Fax:
519-763-2378.
Electronic
mail
address:
Edwardsa@
fhs.McMaster.ca.
t
Dedicated
to
the
memory
of
Dunja
Grbi-Galic
(1950
to
1993).
323
nogenic
toluene
degradation
by
direct
HPLC
analysis
of
the
culture
medium,
but
no
metabolites
were
detected.
We
then
conducted
a
simultaneous
adaptation
experiment
(11)
to
suggest
possible
intermediates.
Each
of
eight
compounds
(p-,
m-
and
o-cresol;
benzyl
alcohol;
benzaldehyde;
benzo-
ate;
phenol;
and
methylcyclohexane),
which
are
good
can-
didates
for
intermediates,
was
fed
simultaneously
with
tolu-
ene
to
the
methanogenic
culture.
This
experiment
is
based
on
the
prediction
that
a
true
intermediate
in
the
catabolic
reaction
would
(i)
be
degraded
without
a
lag
and
(ii)
inhibit
degradation
of
the
substrate
(i.e.,
toluene).
In
an
anaerobic
chamber
(Coy
Laboratory
Products,
Ann
Arbor,
Mich.),
18
16-ml
glass
vials
were
each
filled
with
10
ml
of
active
methanogenic
culture.
The
test
substances
(except
methyl-
cyclohexane)
were
added
individually
to
these
vials
from
aqueous
stock
solutions
to
a
final
concentration
of
100
,uM.
Methylcyclohexane
(100
,uM)
was
added
as
a
neat
solution
with
a
1-,ul
syringe.
All
chemicals
were
purchased
from
Sigma
(St.
Louis,
Mo.)
or
Aldrich
(Milwaukee,
Wis.)
and
were
greater
than
99.9%
pure.
Duplicate
vials
were
prepared
per
test
compound.
The
vials
were
sealed
with
Mininert
screw
caps
(Alltech
Associates,
Inc.
Deerfield,
Ill.)
and
amended
with
neat
toluene
to
a
final
concentration
of
100
,uM
(0.15
RI
per
vial).
Two
control
vials
were
amended
with
toluene
only.
The
vials
were
incubated
at
35°C
in
an
anaer-
obic
chamber.
The
initial
rates
of
degradation
of
toluene
and
of
the
added
test
substances
were
calculated
from
the
initial
concentration
versus
time
data
(first
4
days).
Toluene
and
methylcyclohexane
concentrations
were
measured
by
head-
space
analysis
with
a
gas
chromatograph
(GC)
equipped
with
a
photoionization
detector
(5).
The
concentrations
of
the
remaining
compounds
were
measured
by
HPLC.
A
0.5-ml
sample
was
removed
from
the
vial
with
a
sterile,
disposable
syringe
and
placed
in
a
2-ml
screw-cap
vial.
The
vial
was
centrifuged
for
15
min
at
6,000
x
g.
The
supernatant
was
injected
onto
the
HPLC
column
(described
below)
via
a
100-,u
sample
loop.
The
results
of
the
simultaneous
adapta-
tion
experiment
are
shown
in
Table
1.
m-Cresol,
phenol,
and
methylcyclohexane
were
not
degraded
by
this
culture.
Me-
Vol.
60,
No.
1

APPL.
ENVIRON.
MICROBIOL.
TABLE
1.
Effects
of
potential
toluene-degradative
intermediates
Initial
rate
of
degradation
(pVM/day)"
Potential
substratea
Toluene
Test
substance
Toluene
alone
10.5
±
0.7
None
added
Toluene
+
benzyl
alcohol
2.0
±
3.2
10.0
±
2.0
Toluene
+
benzaldehyde
7.3
+
2.9
8.8
±
1.5
Toluene
+
benzoate
6.5
±
3.1
5.7
±
0.9
Toluene
+
p-cresol
10.3
±
1.3
3.8
±
3.1
Toluene
+
o-cresol
10.6
±
1.2
0
(degraded
after
lag)
Toluene
+
m-cresol
9.6
±
0.4
0
Toluene
+
phenol
10.7
±
0.6
0
Toluene
+
methylcyclohexane
5.7
±
4.6
0
a
Toluene
and
the
test
substance
were
added
at
an
initial
concentration
of
100
,uM.
b
Data
are
the
means
of
duplicates
+
standard
deviations.
thylcyclohexane
appeared
to
have
a
partially
inhibitory
effect
on
toluene
degradation.
Degradation
of
o-cresol
began
eventually,
after
a
lag
of
about
1
week.
Benzyl
alcohol,
benzaldehyde,
benzoate,
and
p-cresol
were
all
degraded
by
the
culture
without
a
lag.
Benzyl
alcohol
was
an
effective
inhibitor
of
toluene
degradation
(Table
1).
In
addition,
ben-
zoic
acid
was
observed
to
transiently
accumulate
in
cultures
fed
either
benzyl
alcohol
or
benzaldehyde
(data
not
shown).
The
data
from
the
simultaneous
adaptation
experiments
identified
benzyl
alcohol,
benzaldehyde,
benzoate,
and
p-cresol
as
possible
intermediates.
Because
the
simulta-
neous
adaptation
method
is
indirect
and
based
on
question-
able
assumptions,
we
sought
a
more
direct
method
to
confirm
these
metabolites
or
to
identify
other
metabolites.
To
overcome
the
problem
of
the
low
concentrations
in-
volved,
we
decided
to
take
advantage
of
the
sensitivity
of
radioactive
analyses,
and
thus
a
method
for
assaying
radio-
active
products
was
sought.
The
initial
intent
was
to
detect
radioactive
metabolites
by
direct
injection
of
culture
fluid
onto
an
HPLC
coupled
to
UV
and
radioactivity
detectors
in
series.
HPLC
and
radioactivity
detector.
The
HPLC
system
con-
sisted
of
a
series
3B
pump
(Perkin-Elmer,
Norwalk,
Conn.),
a
Rheodyne
injector
(0.5
ml
loop),
a
C18
reversed-phase
column
(inside
diameter,
250
mm
by
4.6
mm)
(Absorbo-
sphere
HS;
Alltech),
a
Perkin-Elmer
model
240
diode
array
spectrometer
(UV
detector),
and
a
flow-through
radioactiv-
ity
detector
(1-RAM,
model
1;
IN/US
Systems,
Inc.,
Tampa,
Fla.).
The
latter
was
connected
in
series
after
the
UV
detector.
The
signals
from
the
radioactivity
and
UV
detectors
were
processed
with
software
from
IN/US.
The
UV
detector
was
also
connected
to
a
Nelson
system
inter-
face
(Perkin-Elmer)
and
to
a
plotter
for
displaying
UV
spectral
information.
With
this
system,
it
was
possible
to
obtain
simultaneous
UV
and
radioactive
traces
for
the
peaks
eluting
from
the
column.
The
UV
detector
also
recorded
the
complete
UV
spectrum
for
each
peak
detected.
Two
mobile
phases
were
used:
mobile
phase
A
was
50
mM
phosphate
buffer
(pH
3),
and
mobile
phase
B
was
100%
acetonitrile.
Potential
metabolites
and
toluene
were
eluted
from
the
column
with
the
following
gradient:
80%
A-20%
B,
isocratic
for
52
min,
linear
gradient
to
35%
A-65%
B
over
a
period
of
20
min,
and
35%
A-65%
B,
hold
for
10
min.
The
proportion
of
acetonitrile
was
kept
below
65%
to
preclude
precipitation
of
the
phosphate
buffer.
The
mobile
phase
flow
rate
was
1
ml/min.
The
radioactivity
detector
was
equipped
with
a
1-ml
liquid
flow
cell.
Scintillation
fluid
(INFLO
3;
IN/US
Sys-
tems,
Inc.)
was
mixed
with
the
effluent
from
the
UV
detector
at
a
ratio
of
3:1
before
going
through
the
radioactivity
detector.
In
preliminary
experiments,
cultures
were
fed
radiola-
beled
toluene,
and
the
culture
fluid
was
analyzed
for
radio-
labeled
products
by
HPLC;
no
significant
radioactive
peaks
were
detected,
and
peaks
that
might
elute
with
low
retention
times
were
swamped
by
a
huge
peak
corresponding
to
labeled
dissolved
carbonate
species.
Furthermore,
metabo-
lites
that
may
have
remained
inside
or
attached
to
cells
were
not
recovered
by
this
method,
since
cells
were
removed
by
centrifugation
before
the
sample
was
injected
onto
the
HPLC
column.
An
ether
extraction
procedure
that
solved
many
of
these
problems
was
developed.
By
extraction
into
ether,
the
dissolved
CO2
peak
disappeared;
the
cells
were
lysed;
and
large
aqueous
volumes
could
be
extracted,
con-
centrated,
and
injected
at
one
time,
increasing
the
sensitiv-
ity.
Because
ether
is
immiscible
with
the
HPLC
mobile
phase,
the
concentrated
ether
extract
had
to
be
extracted
back
into
a
water-miscible
phase.
Methanol
was
added
just
before
the
last
of
the
ether
evaporated
to
increase
extraction
efficiency.
The
experimental
details
of
this
extraction
proce-
dure
are
described
below.
A
selective
extraction
of
nonvol-
atile,
water-soluble
compounds
resulted;
this
category
of
compounds
likely
includes
intermediates
of
toluene
degrada-
tion.
With
this
extraction-concentration
scheme,
it
was
necessary
to
begin
with
radiolabeled
toluene
free
of
contam-
inating
radioactive
species.
The
purchased
labeled
toluene
was
highly
contaminated
with
labeled
nonvolatile
compo-
nents.
These
contaminants
were
not
only
potential
interme-
diates
in
the
metabolism
of
toluene,
but
they
were
also
present
in
concentrations
representing
1
to
2%
of
the
label
in
toluene.
Therefore,
['4C]toluene
was
further
purified
by
a
purge
and
trap
method.
Five
hundred
microcuries
of
toluene
(1
,uCi/,umol)
was
transferred
into
5
ml
of
water,
to
which
0.5
ml
of
2
N
NaOH
had
been
added.
This
solution
was
purged
with
N2
and
trapped
in
a
600-,ul
reacti-vial
(Alltech
Associ-
ates,
Inc.)
containing
100
,ul
of
methanol.
The
reacti-vial
was
cooled
with
an
isopropanol-dry
ice
mixture.
The
toluene
was
purged
slowly
for
about
1
h.
Ninety
percent
of
the
toluene
was
recovered
in
the
methanol,
giving
a
final
activity
of
about
3.9
,uCi/,ul
of
toluene-methanol
mixture.
The
radio-
chemical
purity
of
toluene
was
thus
increased
from
98%
to
99.99%.
Experiments
with
radiolabeled
toluene
were
conducted
in
a
series
of
40-ml
vials
sealed
with
Mininert
valves.
Twenty
milliliters
of
active
toluene-degrading
methanogenic
culture
was
added
to
each
vial
inside
an
anaerobic
chamber.
To
add
the
radiolabel,
the
vials
were
removed
from
the
anaerobic
chamber
and
placed
in
a
fume
hood.
Depending
on
the
experiment,
2
to
19
p,Ci
of
purified
[14C]toluene
was
injected
into
each
vial
through
the
Mininert
valve
with
a
10-pI
syringe.
The
vials
were
immediately
placed
inside
an
anaer-
obic
jar
that
was
continuously
flushed
with
N2.
Mininert
valves
provided
an
excellent
seal
for
organic
compounds
such
as
toluene
but
were
quite
leaky
for
gases
(H2,
02,
and
C02).
Therefore,
the
vials
were
incubated
inside
an
anaero-
bic
jar
in
a
fume
hood
(rather
than
in
an
anaerobic
chamber).
The
anaerobic
jar
was
continuously
flushed
with
nitrogen
to
prevent
oxygen
from
diffusing
into
the
jar
and
into
the
sample
vials.
During
the
procedure
of
removing
the
vials
from
the
anaerobic
chamber,
spiking
them
with
radiolabeled
toluene,
and
transferring
them
to
a
N2-flushed
anaerobic
jar,
none
of
the
vials
became
contaminated
with
oxygen
(oxygen
would
have
caused
the
vials
to
turn
pink
because
the
medium
contained
the
redox
indicator
resazurin).
Description
of
the
extraction
method
for
HPLC
analysis
of
324
NOTES

NOTES
325
radiolabeled
compounds.
Vials
were
removed
from
the
anaerobic
jar,
and
the
culture
fluid
was
extracted
following
the
steps
outlined
below
(all
steps
were
performed
in
a
fume
hood).
For
the
first
extraction
(at
neutral
pH),
10
ml
of
diethyl
ether
was
injected
directly
into
the
sample
vial
with
a
glass
syringe
equipped
with
a
22-gauge,
2.5-in.
(ca.
6.4-cm)
stainless
steel
needle.
The
septum
was
first
removed
from
the
Mininert
valve
of
the
sample
vial
to
avoid
a
buildup
of
pressure.
The
sample
vial
was
shaken
vigorously
by
hand
for
3
min
and
vented
by
using
the
Mininert
valve,
and
the
mixture
was
allowed
to
settle
for
about
30
min.
With
the
same
glass syringe,
the
ether
phase
was
transferred
to
a
clean
40-ml
vial
(containing
150
RI
of
phosphate
buffer
[pH
3])
and
capped
with
a
Teflon-lined
screw-cap.
For
the
second
extraction
(at
pH
2),
the
glass
syringe
was
re-inserted
into
the
sample
vial
(without
the
plunger),
400
RI
of
6
N
HCI
was
added
through
the
syringe
barrel
to
acidify
the
sample,
and
then
10
ml
of
clean
ether
was
added.
The
syringe
contents
were
injected
into
the
sample
vial.
The
samples
were
acidi-
fied
to
protonate
organic
acids
such
as
benzoate,
thus
rendering
them
more
soluble
in
ether.
The
sample
vial
was
shaken
vigorously
by
hand
for
3
min
and
vented
by
using
the
Mininert
valve,
and
the
mixture
was
allowed
to
settle
for
about
30
min
(the
pH
was
monitored
with
pH
paper).
Again,
with
the
glass
syringe,
the
second
ether
phase
was
trans-
ferred
to
the
40-ml
vial
containing
the
first
ether
phase
and
capped.
A
final
10
ml
of
diethyl
ether
was
added
to
the
sample
vial,
and
a
third
extraction
was
completed
as
de-
scribed
for
the
second
extraction.
The
combined
ether
extracts
totaled
about
30
ml.
The
combined
ether
extracts
were
evaporated
under
a
stream
of
nitrogen
while
the
vial
was
immersed
in
a
warm
water
bath
(35°C).
Since
the
vial
originally
contained
150
RI
of
phosphate
buffer
and
accumu-
lated
water
from
the
ether,
the
final
aqueous
volume
was
around
200
pul.
When
the
volume
of
ether
left
was
less
than
the
aqueous
volume
(as
determined
by
visual
inspection),
100
,u1
of
methanol
was
added
to
the
sample,
and
then
the
remaining
ether
was
completely
evaporated.
The
remaining
aqueous
extract
(approximately
300
,ul)
thus
recovered
was
ready
to
be
injected
onto
the
HPLC
column.
Samples
containing
particulate
material
were
clarified
by
centrifuga-
tion
before
injection.
Typically,
about
half
the
sample
was
injected
onto
the
HPLC
column,
and
the
other
half
was
saved
for
possible
analysis
by
gas
chromatography-mass
spectrometry.
Initially,
various
surrogate
standards
were
added
to
the
vials
to
monitor
the
extractions;
however,
it
was
found
that
the
resazurin
(or
a
reduction
product
of
resazurin)
present
in
the
medium
eluted
at
a
convenient
retention
time
in
the
UV
chromatogram
and
could
serve
as
a
surrogate
standard.
On
the
basis
of
extractions
with
standard
solutions
of
benzoate,
benzaldehyde,
cresols,
phenols,
and
reduced
resazurin,
the
extraction
efficiency
ranged
from
38
to
55%.
Detection
limit.
The
sensitivity
of
the
radioactivity
detec-
tor
depends,
in
part,
on
the
size
of
the
flow
cell:
the
larger
the
cell,
the
higher
the
sensitivity,
but
the
lower
the
resolu-
tion.
With
a
1-ml
flow
cell
(as
used
in
our
experiments),
a
peak
of
150
cpm
or
2.5
Bq
(about
five
times
noise
level)
could
be
detected.
On
the
basis
of
this
detection
limit
and
a
substrate
specific
activity
of
1
,uCi/,umol,
we
could
detect
labeled
compounds
as
dilute
as
10
nM
in
a
20-ml
culture
fluid;
this
is
comparable
to
the
detection
limits
of
gas
chromatography-mass
spectrometry.
The
sensitivity
could
be
increased
further
by
using
a
substrate
with
higher
specific
activity.
Even
with
the
sensitivity
of
the
overall
extraction-HPLC
A10.
0
B
0
2
0
Bio
1
cio
0
.-A
0
-8
-9
I
IV
I
j.-
0
0
0
43
m
m
.............
4
)
10
i
00
.G
t
w
As
E
I R
r
A
n
30
",.
..-.
...-
IV
....
....
t--@el
...
A
-1
......
..
0
8
16
24
32
40
48
56
64 72
80
Retention
Time
(minutes)
FIG.
1.
Three
chromatograms
from
HPLC
analysis
showing
radioactivity
detector
output
from
methanogenic
culture
amended
with
radiolabeled
toluene
and
unlabeled
benzoate
(A),
with
radiola-
beled
toluene
and
unlabeled
benzyl
alcohol
(B),
and
with
radiola-
beled
toluene
only
(C).
method,
we
did
not
detect
any
significant
radioactive
prod-
ucts
in
cultures
fed
labeled
toluene,
even
after
increasing
the
amount
of
['4C]toluene
added
per
vial
from
2
to
19
p,Ci.
This
indicated
that
metabolites
did
not
normally
accumulate
to
concentrations
higher
than
the
detection
limit
of
10
nM.
To
increase
the
concentration
of
labeled
metabolites,
we
ap-
plied
the
method
of
isotope
trapping.
Cultures
actively
degrading
labeled
toluene
were
challenged
with
a
large
dose
of
an
unlabeled
compound
(i.e.,
a
potential
intermediate)
to
trap
radiolabel
in
upstream
metabolites.
Six
40-ml
glass
vials
filled
with
20
ml
of
active
methanogenic
culture
were
spiked
with
19
p,Ci
of
labeled
toluene
(5
pll
of
purified
labeled
toluene-methanol
mixture
per
vial).
After
3
days
of
incuba-
tion,
approximately
35%
of
the
toluene
was
degraded
(as
determined
by
liquid
scintillation
counting
of
a
small
aliquot
from
a
vial).
At
this
time,
five
vials
were
quickly
removed
from
the
anaerobic
jar
and
spiked
with
200
p,M
of
either
benzyl
alcohol,
benzoate,p-cresol,
m-cresol
or
o-cresol.
The
sixth
vial
was
not
amended.
Four
days
later,
samples
from
all
the
vials
were
extracted
and
analyzed.
Figure
1
shows
the
chromatograms
obtained
from
the
VOL.
60,
1994

APPL.
ENVIRON.
MICROBIOL.
TABLE
2.
Radioactivity
recovered
in
toluene
metabolites
after
addition
of
isotope-trapping
compounds
to
a
["4C]toluene-degrading
methanogenic
culture
Radiolabeled
Retention
time
Radioactivity
recovered
(cpm)b
after
addition
of
unlabeled
isotope-trapping
compounds
metabolitea
(min)
None
Benzyl
alcohol
Benzoate
p-Cresol
o-Cresol
m-Cresol
Benzyl
alcohol
13.4
0
1,590
480
160
0
0
Benzaldehyde
32.7
0
4,730
1,040
0 0
0
Benzoate
23.5
0
1,090
5,200
100
00
p-Cresol
38.5
0
1,620
350
590
250
320
Unknown
42.8
1,282
1,860
800
1,020
200
230
a
Metabolite
corresponding
to
the
HPLC
peak.
b
In
these
experiments,
150
cpm
corresponded
to
approximately
10
nM.
radioactivity
detector
after
HPLC
analysis
of
samples
from
three
vials
from
the
isotope
trapping
experiment:
one
that
had
been
amended
with
benzoate
as
a
trapping
agent,
one
that
had
been
amended
with
benzyl
alcohol,
and
finally
one
that
had
no
amendments.
All
the
radioactivity
recovered
in
the
various
peaks
originated
from
labeled
toluene.
Peaks
of
radioactivity
corresponding
to
benzoate,
benzyl
alcohol,
benzaldehyde,
p-cresol,
toluene,
and
acetate
were
identified
by
matching
retention
times
and
UV
spectra
with
those
of
authentic
standards
(Fig.
1).
Authentic
standards
of
many
potential
intermediates,
including
p-hydroxybenzoate,
p-
and
o-toluate,
m-
and
o-cresol,
cyclohexane
carboxylic
acid,
phthalate,
phenylacetate,
and
phenylpropionate,
were
ana-
lyzed
for
comparison
with
unknown
peaks.
None
of
these
compounds
corresponded
to
the
unknown
peaks
of
the
chromatograms.
The
amount
of
radioactivity
found
in
each
peak
(normalized
on
the
basis
of
the
area
of
the
resazurin
peak
in
the
UV
chromatogram)
is
shown
in
Table
2.
When
no
trapping
agent
was
added
to
the
culture,
very
few
peaks
could
be
detected
in
the
region
of
interest
(i.e.,
the
region
spanning
retention
times
from
9
to
50
min,
at
which
all
standards
of
potential
aromatic
intermediates
eluted).
Simi-
larly,
o-
and
m-cresol
did
not
cause
a
significant
buildup
of
radioactive
metabolites
(Table
2).
However,
benzyl
alcohol
and
benzoate
were
very
effective
trapping
agents.
Both
compounds
caused
significant
increases
in
the
radioactivity
detected
in
the
peaks
corresponding
to
benzyl
alcohol,
benzaldehyde,
benzoate,
and
p-cresol
(Table
2).
p-Cresol
was
less
effective,
causing
a
barely
detectable
buildup
of
benzyl
alcohol
and
a
small
accumulation
of
labeledp-cresol.
An
unidentified
radiolabeled
peak
eluting
at
about
42.8
min
was
detected
in
all
the
samples;
it
appeared
to
accumulate
in
the
culture
fluid.
The
combination
of
isotope
trapping
and
radiodetection
used
in
this
study
has
enabled
the
identification
of
several
compounds
as
possible
intermediates
in
the
methanogenic
degradation
of
toluene.
Some
or
all
of
the
compounds
identified,
namely
benzoic
acid,
benzyl
alcohol,
benzalde-
hyde,
and
p-cresol,
have
previously
been
implicated
in
anaerobic
toluene
degradation
(1-3,
6-10,
12).
To
determine
the
metabolic
relationship
between
the
various
intermedi-
ates,
to
distinguish
between
major
and
minor
pathways
of
degradation,
and
to
identify
possible
dead-end
metabolites,
the
accumulation
of
label
in
the
various
compounds
during
isotope
trapping
experiments
must
be
determined
as
a
func-
tion
of
time,
perhaps
by
using
a
pulse-chase
procedure.
To
test
the
general
applicability
of
our
method,
we
used
this
radioactivity
detection
system
to
study
the
degradation
of
radiolabeled
toluene
in
a
sulfate-reducing
toluene-degrad-
ing
culture
(5).
To
our
surprise,
even
in
the
absence
of
trapping
compounds,
several
intermediates
were
detected.
Though
most
of
the
intermediates
remain
uncharacterized
at
present
(only
benzoate
and
benzylsuccinate
have been
iden-
tified
[3a]),
their
abundance
was
50-
to
100-fold
higher
than
the
abundance
of
those
observed
in
the
methanogenic
cul-
ture.
These
studies
indicate
that
there
are
significant
varia-
tions
between
cultures
in
the
relative
accumulation
of
the
catabolic
intermediates
and
therefore
suggest
that
a
judi-
cious
choice
of
the
culture
may
greatly
aid
in
the
identifica-
tion
of
the
elusive
initial
anaerobic
biotransformation
inter-
mediates.
This
project
was
supported
through
the
U.S. Environmental
Protection
Agency-supported
Western
Region
Hazardous
Sub-
stance
Research
Center
at
Stanford
University,
grants
from
the
U.S.
Air
Force
(AFOSR
88-0351),
U.S.
Environmental
Protection
Agency
(EPA
R
815252-01-0),
and
NSF
(NSF
CES
8813958)
awarded
to
D.G.-G.
and
a
scholarship
from
the
Quebec
government
(F.C.A.R.)
awarded
to
E.A.E.
A.M.E.
is
a
Research
Scholar
of
the
Medical
Research
Council
of
Canada.
We
thank
Harry
Beller,
Harry
Ball,
and
Ned
Black
for
valuable
discussions
and
two
anonymous
reviewers
for
critical
review
of
the
manuscript.
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NOTES

VOL.
60,
1994
NOTES
327
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