Anaerobic degradation of toluene and o-xylene by a methanogenic consortium

Abstract

Toluene and o-xylene were completely mineralized to stoichiometric amounts of carbon dioxide, methane, and biomass by aquifer-derived microorganisms under strictly anaerobic conditions. The source of the inoculum was creosote-contaminated sediment from Pensacola, Fla. The adaptation periods before the onset of degradation were long (100 to 120 days for toluene degradation and 200 to 255 days for o-xylene). Successive transfers of the toluene- and o-xylene-degrading cultures remained active. Cell density in the cultures progressively increased over 2 to 3 years to stabilize at approximately 10(9) cells per ml. Degradation of toluene and o-xylene in stable mixed methanogenic cultures followed Monod kinetics, with inhibition noted at substrate concentrations above about 700 microM for o-xylene and 1,800 microM for toluene. The cultures degraded toluene or o-xylene but did not degrade m-xylene, p-xylene, benzene, ethylbenzene, or naphthalene. The degradative activity was retained after pasteurization or after starvation for 1 year. Degradation of toluene and o-xylene was inhibited by the alternate electron acceptors oxygen, nitrate, and sulfate. Degradation was also inhibited by the addition of preferred substrates such as acetate, H2, propionate, methanol, acetone, glucose, amino acids, fatty acids, peptone, and yeast extract. These data suggest that the presence of natural organic substrates or contaminants may inhibit anaerobic degradation of pollutants such as toluene and o-xylene at contaminated sites.

Full text

APPLIED
AND
ENVIRONMENTAL
MICROBIOLOGY,
Jan.
1994,
p.
313-322
0099-2240/94/$04.00+0
Copyright
©
1994,
American
Society
for
Microbiology
Anaerobic
Degradation
of
Toluene
and
o-Xylene
by
a
Methanogenic
Consortiumt
ELIZABETH
A.
EDWARDS*
AND
DUNJA
GRBIC-GALIC
Environmental
Engineering
and
Science,
Department
of
Civil
Engineering,
Stanford
University,
Stanford,
California
94305-4020
Received
19
May
1993/Accepted
2
November
1993
Toluene
and
o-xylene
were
completely
mineralized
to
stoichiometric
amounts
of
carbon
dioxide,
methane,
and
biomass
by
aquifer-derived
microorganisms
under
strictly
anaerobic
conditions.
The
source
of
the
inoculum
was
creosote-contaminated
sediment
from
Pensacola,
Fla.
The
adaptation
periods
before
the
onset
of
degradation
were
long
(100
to
120
days
for
toluene
degradation
and
200
to
255
days
for
o-xylene).
Successive
transfers
of
the
toluene-
and
o-xylene-degrading
cultures
remained
active.
Cell
density
in
the
cultures
progressively
increased
over
2
to
3
years
to
stabilize
at
approximately
109
cells
per
ml.
Degradation
of
toluene
and
o-xylene
in
stable
mixed
methanogenic
cultures
followed
Monod
kinetics,
with
inhibition
noted
at
substrate
concentrations
above
about
700
FM
for
o-xylene
and
1,800
FM
for
toluene.
The
cultures
degraded
toluene
or
o-xylene
but
did
not
degrade
m-xylene,
p-xylene,
benzene,
ethylbenzene,
or
naphthalene.
The
degradative
activity
was
retained
after
pasteurization
or
after
starvation
for
1
year.
Degradation
of
toluene
and
o-xylene
was
inhibited
by
the
alternate
electron
acceptors
oxygen,
nitrate,
and
sulfate.
Degradation
was
also
inhibited
by
the
addition
of
preferred
substrates
such
as
acetate,
H2,
propionate,
methanol,
acetone,
glucose,
amino
acids,
fatty
acids,
peptone,
and
yeast
extract.
These
data
suggest
that
the
presence
of
natural
organic
substrates
or
cocontaminants
may
inhibit
anaerobic
degradation
of
pollutants
such
as
toluene
and
o-xylene
at
contaminated
sites.
Soil,
sediment,
and
groundwater
are
frequently
contami-
nated
with
petroleum
products
as a
result
of
leaks
in
under-
ground
storage
tanks,
improper
disposal
techniques,
and
inadvertent
spills.
Of
the
many
constituents
of
petroleum,
the
nonoxygenated,
homocyclic
aromatic
compounds
(in-
cluding
benzene,
toluene,
xylenes,
and
ethylbenzene)
are
of
particular
concern
because
they
are
confirmed
or
suspected
carcinogens,
even
at
very
low
concentrations
(6).
These
compounds
are
relatively
water
soluble
compared
with
other
components
of
petroleum
and
thus
frequently
migrate
through
groundwater
systems
to
contaminate
drinking
water
supplies
far
removed
from
the
actual
spill
(5,
29).
The
microbial
degradation
of
compounds
such
as
toluene
and
benzene
under
aerobic
conditions
has
been
studied
in
great
detail
(13,
30);
in
contrast,
the
fate
of
homocyclic
aromatic
compounds
in
anaerobic
environments
is
very
poorly
under-
stood.
Until
the
mid-1980s,
it
was
generally
believed
that
monoaromatic
compounds
were
recalcitrant
to
degradation
under
anaerobic
conditions.
Certain
monoaromatic
hydro-
carbons,
most
frequently
toluene,
have
since
been
shown
to
be
degraded
by
microorganisms
under
denitrifying
(8,
11,
20,
21,
25,
36),
iron-reducing
(22,
23),
sulfate-reducing
(2,
10,
17)
and
methanogenic
(16,
32,
33,
34)
conditions.
We
report
here
the
enrichment
and
maintenance
of
a
mixed
culture
derived
from
contaminated
aquifer
sediments
that
specifically
de-
grades
toluene
and
o-xylene
under
methanogenic
conditions.
The
culture
has
been
maintained
with
toluene
or
o-xylene
as
the
sole
sources
of
carbon
and
energy
for
more
than
3
years.
The
growth
and
degradation
kinetics
and
the
effects
of
alternate
electron
acceptors
and
substrates
are
described
*
Corresponding
author.
Present
address:
Beak
Consultants
Ltd.,
42
Arrow
Rd.,
Guelph,
Ontario,
Canada,
NlK-1S6.
Phone:
519-
763-2325
ext.
236.
Fax:
519-763-2378.
Electronic
mail
address:
Edwardsa@fhs.McMaster.ca.
t
Dedicated
to
the
memory
of
Dunja
Grbic-Galic
(1950-1993).
313
and
discussed
in
this
paper.
The
results
of
a
study
on
the
metabolites
of
toluene
degradation
by
this
culture
are
pre-
sented
in
a
separate
paper
(9).
MATERIALS
AND
METHODS
Aquifer
material.
Aquifer
solids
from
Pensacola,
Fla.,
were
provided
by
E.
M.
Godsy
(U.S.
Geological
Survey,
Menlo
Park,
Calif.).
The
Pensacola
aquifer
consists
of
fine-
to-coarse
sand
deposits,
interrupted
by
discontinuous
silts
and
clay.
The
upper
30
m
of
the
aquifer
is
contaminated
by
creosote
and
pentachlorophenol.
The
samples
were
obtained
from
an
actively
methanogenic
sandy
zone
of
the
aquifer,
downgradient
from
the
contamination
source,
from
a
depth
of
approximately
6
m.
The
groundwater
at
this
depth
con-
tained
tens
of
milligrams
of
nitrogen
heterocycles,
simple
polyaromatic
hydrocarbons,
and
phenols
per
liter.
The
in-
digenous
microorganisms
have
been
shown
to
anaerobically
degrade
aromatic
and
heterocyclic
constituents
of
the
water-
soluble
fraction
of
creosote
(14,
15).
Aseptic
sampling
was
performed,
and
the
aquifer
core
was
stored
(at
4°C)
in
sterile
sealed
containers
previously
flushed
with
argon
(14).
Medium.
A
medium
designed
to
support
methanogenic
bacteria
that
had
the
following
constituents
per
liter
of
deionized
water
was
prepared:
10
ml
of
phosphate
buffer
(27.2
g
of
KH2PO4
per
liter,
34.8
g
of
K2HPO4
per
liter),
10
ml
of
salt
solution
(53.5
g
of
NH4Cl
per
liter,
7.0
g
of
CaCl2.
6H20
per
liter,
2.0
g
of
FeCl2
4H20
per
liter),
2
ml
of
trace
mineral
solution
[0.3
g
of
H3BO3
per
liter,
0.1
g
of
ZnCl2
per
liter,
0.75
g
of
NiCl2
6H20
per
liter,
1.0
g
of
MnCl2.
4H20
per
liter,
0.1
g
of
CuCl2
2H20
per
liter,
1.5
g
of
CoCl2
6H20
per
liter,
0.02
g
of
Na2SeO3
per
liter,
0.1
g
of
A12(SO4)3
16H20
per
liter,
1
ml
of
H2SO4
per
liter],
2
ml
of
MgSO4.
7H20
solution
(62.5
g/liter),
1
ml
of
redox
indicator
stock
solution
(1
g
of
resazurin
per
liter),
10
ml
of
saturated
bicarbonate
solution
(260
g
of
NaHCO3
per
liter),
Vol.
60,
No.
1

314
EDWARDS
AND
GRBIC-GALIC
10
ml
of
filter-sterilized
vitamin
stock
solution
(0.02
g
of
biotin
per
liter,
0.02
g
of
folic
acid
per
liter,
0.1
g
of
pyridoxine
hydrochloride
per
liter,
0.05
g
of
riboflavin
per
liter,
0.05
g
of
thiamine
per
liter,
0.05
g
of
nicotinic
acid
per
liter,
0.05
g
of
pantothenic
acid
per
liter,
0.05
g
of
p-amino-
benzoic
acid
per
liter,
0.05
g
of
cyanocobalamin
per
liter,
0.05
g
of
thioctic
acid
per
liter,
1
g
of
mercaptoethanesulfonic
acid
[coenzyme
M]
per
liter),
and
10
ml
of
an
amorphous
ferrous
sulfide
solution
[39.2
g
of
(NH4)2Fe(SO4)2
6H20
per
liter,
24.0
g
of
Na2S
9H20
per
liter]
that
had
been
washed
three
times
with
deionized
water
to
remove
free
sulfide
(4).
The
vitamins,
bicarbonate,
and
ferrous
sulfide
were
added
from
sterile
stock
solutions
after
the
medium
had
been
autoclaved
and
cooled
while
being
gassed
with
N2-CO2
(80:20
[vol/vol]).
The
pH
of
the
medium
was
usually
between
6.8
and
7.0.
In
the
experiments
designed
to
test
the
effect
of
pH
on
degradation,
the
pH
of
the
medium
was
adjusted
to
6
with
1
N
HCl
or
to
8
with
1
N
NaOH.
Chemicals.
Chemicals
were
purchased
from
Sigma
Chem-
ical
Co.
(St.
Louis,
Mo.)
or
Aldrich
Chemicals
(Milwaukee,
Wis.)
and
were
greater
than
99.9%
pure.
[methyl-14C]tolu-
ene,
[ring-14C]toluene,
and
[methyl-
4C]o-xylene
were
also
purchased
from
Sigma
and
had
specific
activities
of
4.9,
9.5,
and
10.9
mCi/mmol,
respectively.
The
radiochemical
purity
of
these
compounds
was
about
99%.
Microcosms.
Microcosms
were
prepared
in
250-ml
(8
oz.)
screw-cap
bottles
and
sealed
with
Mininert
valves
(Alltech
Associates,
Inc.,
Deerfield,
Ill.).
The
bottles
and
caps
were
acid
washed,
sterilized,
and
brought
into
an
anaerobic
glove
box
(Coy
Laboratory
Products,
Ann
Arbor,
Mich.).
All
glassware
was
preincubated
in
the
glove
box
for
at
least
1
day
to
remove
all
traces
of
oxygen.
Aquifer
material
(100
g)
followed
by
100
ml
of
medium
was
added
to
250-ml
bottles.
Five
groups
of four
microcosms
were
prepared.
Each
group
consisted
of
a
chemical
control
microcosm
(medium
plus
substrates,
no
sediment),
an
autoclaved
biological
control
microcosm
(autoclaved
sediment,
medium
plus
substrates),
and
two
replicate
test
microcosms.
The
sediment
in
sterile
control
bottles
was
autoclaved
for
20
min
at
121°C
on
3
consecutive
days
before
medium
was
added
to
these
bottles.
Each
group
was
designed
to
test
a
different
combination
of
substrates
and
concentrations.
The
first
three
groups
were
amended
with
a
mixture
of
substituted
monoaromatic
com-
pounds
(toluene,
o-xylene,
p-xylene,
and
ethylbenzene)
at
either
an
initial
aqueous
concentration
of
40
,uM
for
each
compound
(first
and
second
groups)
or
at
an
initial
concen-
tration
of
320
,uM
for
each
compound
(third
group).
The
microcosms
in
the
second
group
were
additionally
amended
with
460
,uMp-cresol.
The
fourth
and
fifth
groups
of
micro-
cosms
were
amended
with
the
unsubstituted
aromatic
com-
pounds,
benzene
and
naphthalene
(initial
concentrations,
150
and
30
,uM,
respectively),
with
and
without
addition
of
570
,uM
phenol.
The
putative
intermediates,
p-cresol
and
phenol,
were
added
in
some
cases
to
attempt
to
stimulate
growth
of
hydrocarbon-degrading
organisms.
All
manipula-
tions
and
incubations
were
performed
in
an
anaerobic
glove
box
(atmospheric
composition:
85%
N2,
10%
C02,
and
5%
H2).
The
bottles
were
incubated
statically
at
35°C
in
the
dark.
Enrichment
cultures.
After
8
months
of
refeeding
active
microcosms
with
toluene
or
o-xylene
(approximately
once
per
month
with
1.5
to
3.0
RI
of
pure
toluene
or
o-xylene),
primary
enrichment
cultures
were
prepared
by
transferring
sediment
(10
g
[wet
weight])
and
liquid
(30
ml)
from
active
microcosms
to
four
clean,
autoclaved
250-ml
bottles.
These
bottles
were
then
filled
with
170
ml
of
prereduced,
defined
mineral
medium
and
gassed
with
N2-CO2
(80:20
[vol/vol]).
Two
of these
bottles
were
spiked
with
both
toluene
and
o-xylene
(as
pure
compounds)
at
5
mg/liter
(50
,uM)
each.
The
third
was
amended
with
toluene
only
(50
,uM),
and
the
fourth
was
amended
with
o-xylene
only.
Secondary
enrich-
ment
cultures
that
no
longer
contained
aquifer
solids
were
prepared
by
inoculating
fresh
medium
with
some
of
the
liquid
from
primary
enrichment
cultures
(10
to
30%
inocu-
lum).
Stable
suspended
cultures
were
maintained
by
refeed-
ing
weekly
with
toluene,
o-xylene,
or
both
(10
,ul
of
toluene
or
o-xylene
per
200
ml
of
culture)
and
by
replacing
25
to
50%
of
the
shaken
culture
medium
with
fresh
medium
approxi-
mately
once
every
4
months.
Certain
cultures
were
replen-
ished
with
medium
that
had
been
filtered
to
remove
the
black
precipitate,
ferrous
sulfide
(FeS).
After
being
replenished
several
times
with
filtered
medium,
the
culture
medium
was
devoid
of
solid
precipitates
that
interferred
with
techniques
such
as
protein
measurement
and
microscopy.
Mass
balance
determinations.
To
determine
the
amount
of
methane
formed from
the
degradation
of toluene
and
o-xy-
lene,
a
known
amount
of
substrate
was
added
to
200
ml
of
a
freshly
replenished
culture
flushed
with
a
N2-CO2
gas
mix-
ture
(to
remove
traces
of
H2).
The
concentration
of
methane
in
the
headspace
was
measured
after
the
substrate
was
fully
depleted.
To
determine
the
amount
of
CO2
and
nonvolatile
products
formed
during
the
degradation
of
toluene
and
o-xylene,
cultures
were
spiked
with
radiolabeled
substrates
(t14C]toluene
[both
methyl
and
ring
labeled]
or
[methyl-
C]o-xylene),
and
the
14C
activities
in
the
volatile,
nonvol-
atile,
and
CO2
fractions
were
determined
as
degradation
proceeded,
following
the
method
described
in
reference
16.
4C
activity
in
liquid
samples
was
determined
with
a
Tricarb
model
4530
scintillation
spectrometer
(Packard
Instrument
Co.,
Downers
Grove,
Ill.).
Counting
efficiency
corrections
were
made
with
the
external
standard
channels
ratio
method
(1).
Kinetic
experiments.
The
cell
yield
(Y)
was
determined
in
cultures
growing
in
filtered
medium
by
measuring
the
differ-
ence
in
the
protein
content
of
the
culture
before
and
after
the
degradation
of
a
known
amount
of
substrate.
Protein
content
was
determined
by
the
method
of
Bradford
(3)
and
converted
to
cell
mass,
assuming
that
50%
of
the
cell
dry
weight
is
protein.
Substrate
depletion
was
monitored
for
a
series
of
initial
substrate
concentrations
ranging
from
50
p.M
to
2.5
mM.
The
initial
active
biomass
concentration
(X0)
was
estimated
from
cell
counts
and
protein
determinations
for
samples
of
the
culture
taken
at
the
start
of
the
experiment.
To
determine
cell
counts,
10-,ul
samples
were
spread
over
a
1-cm2
area
on
a
microscope
slide.
The
samples
were
heat
fixed,
stained
with
acridine
orange
(0.01%)
for
2
min,
and
then
washed
with
water.
The
cells
were
observed
in
oil
immersion
with
an
epifluorescence
microscope
(Olympus
Optical
Co.
Ltd.,
Tokyo,
Japan)
equipped
with
an
Olympus
FLPL
x
100
objective
lens
and
a
x
10
ocular
lens.
Sixteen
to
twenty
fields
per
sample
were
counted,
and
the
average
cell
count
per
field
was
used
to
calculate
the
total
cell
count,
given
that
the
area
of
the
field
was
1.7
x
10-3
mm2.
The
substrate
depletion
curves
were
fit
to
the
Monod
kinetic
model
(27)
adapted
for
a
volatile
substrate
in
a
two-phase
system.
The
decay
term
(endogenous
respiration)
was
ne-
glected
since
decay
rates
are
relatively
small
in
anaerobic
systems.
Nonlinear
regression
of
the
data
from
substrate
depletion
curves
to
the
Monod
kinetic
model
provided
estimates
of
the
parameters
Ks
(half-saturation
constant)
and
Amax
(maximum
specific
growth
rate).
The
Monod
kinetic
model
for
a
volatile
substrate
is
APPL.
ENvIRON.
MICROBIOL.

METHANOGENIC
DEGRADATION
OF
TOLUENE
AND
o-XYLENE
315
-dM/dt
=
(Lmax/Y)
Xo
VL
[S/(Ks
+
5)],
where
M
is
the
total
mass
of
substrate
in
the
incubation
bottle
at
a
given
time
(in
milligrams),
t
is
time
(in
days),
PUmax
is
the
maximum
specific
growth
rate
per
day,
Y
is
the
yield
(in
milligrams
per
milligrams),
X0
is
the
initial
active
biomass
concentration
(in
milligrams
per
liter),
VL
is
the
liquid
volume
(in
milliliters),
S
is
the
substrate
aqueous
concentration
at
a
given
time
(in
milligrams
per
liter),
and
Ks
is
the
half
saturation
or
affinity
constant
(in
milligrams
per
liter).
The
mass
at
any
given
time
is
related
to
the
liquid
concentration
by
the
relationship
S
=
M/(VL
+
H
VG),
where
H
is
the
dimensionless
Henry's
Law
constant
for
the
substrate
and
VG
is
the
headspace
volume
(in
milliliters).
Pasteurization.
A
culture
(120
ml)
that
had
been
starved
for
1
month
and
another
culture
(120
ml)
that
was
actively
degrading
toluene
were
pasteurized
at
80°C
for
15
min.
The
cultures
were
cooled,
dispensed
into
four
40-ml
vials
each
(30-ml
culture
per
vial),
and
refed
toluene.
Two
of
the
four
vials
from
each
culture
(starved
and
active)
also
received
an
inoculum
of
methanogens
previously
enriched
with
H2
from
the
toluene-degrading
culture
(toluene
was
not
degraded
by
these
methanogens
in
control
experiments).
Toluene
con-
centrations
in
pasteurized
cultures
were
monitored
over
time
in
parallel
with
two
sterile
control
vials
to
assess
the
survival
of
the
members
of
the
culture
following
the
heat
shock
of
pasteurization.
The
malachite
green-staining
procedure
was
used
to
determine
whether
endospores
were
present
(7).
Cell
freezing.
Both
toluene-
and
o-xylene-degrading
stable
cultures
(200
ml
of
each)
were
centrifuged
(6,000
x
g,
45
min)
in
gas-tight
bottles
in
a
Damon/IEC
(Needham,
Mass.)
model
DPR-6000
centrifuge.
Inside
an
anaerobic
chamber,
the
supernatant
was
discarded,
and
the
cell
pellets
were
resuspended
with
1
ml
of
filtered
anaerobic
medium
in
1.5-ml
Eppendorf
tubes.
Seventy
microliters
of
dimethyl
sulfoxide
was
added
to
each
tube.
The
tubes
were
vortexed,
removed
from
the
glove
box,
and
immediately
flash
frozen
in
liquid
nitrogen.
Frozen
cells
were
stored
at
-80°C.
Electron
microscopy.
Toluene-
and
o-xylene-degrading
en-
richment
cultures
which
had
been
transferred
several
times
into
filtered
medium
that
no
longer
contained
FeS
precipitate
were
prepared
for
scanning
electron
microscopy.
The
cul-
tures
(100
ml)
were
centrifuged
(6,000
x
g,
45
min),
and
the
pellets
were
resuspended
in
1
ml
of
phosphate
buffer
(0.1
M,
pH
7.2).
The
cultures
were
fixed
with
glutaraldehyde,
stained
with
osmium
tetroxide
and
uranyl
acetate,
and
dehydrated
in
increasing
strengths
of
ethanol
(18).
The
samples
were
then
dried
with
hexamethyldisilazane
(HMDS;
Polysciences,
Inc.,
Warrington,
Pa.)
and
examined
with
a
Philips
505
scanning
electron
microscope.
Effects
of
alternate
electron
acceptors,
alternate
substrates,
and
inhibiting
substances
on
toluene
degradation.
For
each
experiment,
a
10
to
50%
inoculum
from
a
toluene-degrading
enrichment
culture
was
transferred
to
the
required
number
of
40-ml
vials.
The
volume
was
made
up
to
30
ml
with
fresh
medium,
and
the
vials
were
sealed
with
Mininert
valves.
In
the
experiments
testing
the
effects
of
oxygen
and
nitrate,
cultures
growing
in
medium
devoid
of
FeS
precipitate
were
used.
Oxygen
was
added
by
injecting
air
from
a
syringe
into
the
vials,
and
the
actual
concentration
added
was
calculated
on
the
basis
of
the
volume
of
air
added,
the
Henry's
Law
constant
and
density
of
air,
the
percent
oxygen
in
the
air,
and
the
liquid
and
gas
volumes
in
the
vial.
Nitrate
and
sulfate
were
added
as
NaNO3
and
Na2SO4,
respectively.
Other
substances
tested
as
potentially
stimulating
alternate
sub-
strates
included
acetate,
hydrogen,
glucose,
Casamino
Acids
(commercial
mixture),
sodium
propionate,
a
fatty
acid
mix-
ture
(butyric,
valeric,
and
caproic
[both
n
and
iso]),
yeast
extract,
cysteine-HCI,
and
acetone.
The
toluene-degrading
cultures
were
also
challenged
with
cyclohexane,
carbon
tetrachloride,
and
benzene
to
assess
the
inhibitory
effects
of
these
compounds.
Each
of
these
test
substances
was
added
to
duplicate
cultures
from
neutralized
aqueous
stock
solu-
tions
at
different
concentrations
ranging
over
2
orders
of
magnitude.
Toluene
or
o-xylene
was
also
added
to
these
vials,
at
a
fixed
concentration
of
about
200
,uM.
Analytical
procedures.
Toluene,
xylenes,
ethylbenzene,
benzene,
and
cyclohexane
concentrations
were
measured
by
withdrawing
300
,u
of
headspace
from
sample
bottles
with
a
500-pI
gas-tight
syringe
and
injecting
the
headspace
into
a
Fractovap
2900
series
gas
chromatograph
(Carlo
Erba
Stru-
mentazione,
Milan,
Italy)
equipped
with
a
photoionization
detector
(model
PI-52-02;
10
eV
lamp;
HNU
Systems,
Inc.)
and
a
DB-624
fused
silica
megabore
capillary
column
(inside
diameter,
30
by
0.53
mm;
J&W
Scientific,
Folsom,
Calif.).
The
operating
conditions
for
the
gas
chromatograph-photo-
ionization
detector
were
an
injection
port
temperature
of
240°C,
a
detector
temperature
of
250°C,
helium
carrier
gas
at
a
column
head
pressure
of
0.7
kg/cm2,
helium
make-up
gas
at
a
flow
of
30
ml/min,
an
isothermal
temperature
of
90°C,
and
a
splitless
injection
(split
closed
for
30
s).
The
data
from
the
gas
chromatograph-photoionization
detector
were
collected
and
processed
with
the
Nelson
Analytical,
Inc.,
3000
Series
Chromatography
Data
System.
The
aqueous
concentration
of
aromatic
compounds
in
microcosms
and
enrichments
was
determined
by
comparing
peak
areas
with
those
of
stan-
dards.
Standards
for
headspace
analyses
were
prepared
by
spiking
a
methanolic
stock
solution
of
the
aromatic
com-
pound
into
a
Mininert-sealed
bottle
that
contained
200
ml
of
water.
The
transfer
of
the
stock
solution
was
done
with
a
gas-tight
500-ml
syringe.
The
amount
of
stock
solution
added
to
the
standard
bottle
was
determined
gravimetrically
by
weighing
the
syringe
immediately
before
and
after
spiking.
The
aqueous
concentration
of
aromatic
compounds
in
stan-
dards
was
calculated
by
using
Henry's
Law
constants
ob-
tained
from
reference
24.
Methane
concentrations
were
determined
by
injecting
400
pA
of
headspace
onto
a
Fisher-
Hamilton
gas
partitioner
(model
25V;
Fisher
Scientific,
Pittsburg,
Pa.)
equipped
with
a
thermal
conductivity
de-
tector
and
helium
carrier
gas
(60
ml/min).
Certified
gas
standards
were
used
for
calibration.
p-Cresol,
phenol,
and
naphthalene
were
analyzed
by
high-performance
liquid
chro-
matography
(HPLC)
(Series
400
Liquid
Chromatograph;
Perkin-Elmer
Cetus,
Norwalk,
Conn.)
equipped
with
a
C18
reverse-phase
column
(inside
diameter,
250
mm
by
4.6
mm;
Alltech
Associates)
and
a
1050
Series
Variable
Wavelength
Detector
(Hewlett-Packard,
Avondale,
Pa.).
Samples
were
centrifuged
at
5,000
x
g
for
5
min
to
remove
particulates
before
injection
onto
the
HPLC.
Data
were
collected
and
processed
with
a
SP
4020
Data
Interface
(Spectra-Physics,
Santa
Clara,
Calif.).
The
eluant
was
70%
acetate
buffer
(50
mM,
pH
4.5)
and
30%
methanol
at
a
flow
rate
of
1
ml/min
(isocratic
analysis);
the
detection
wavelength
was
280
nm.
RESULTS
Initial
adaptation
period.
After
long
lag
times,
degradative
activity
was
detected
in
some
of
the
microcosms
amended
with
substituted
monoaromatic
compounds
at
a
low
concen-
tration
(total
concentration,
160
puM).
p-Cresol
degradation
was
complete
in
80
days.
Toluene
and
o-xylene
transforma-
tion
began
in
one
of
the
two
replicates
from
the
group
without
p-cresol
and
in
one
of
the
two
replicates
from
the
VOL.
60,
1994

316
EDWARDS
AND
GRBIC-GALIC
group
supplemented
with
p-cresol.
The
lag
times
before
the
onset
of
degradation
were
100
or
120
days
for
toluene
and
200
or
255
days
for
o-xylene
(with
and
without
p-cresol,
respectively).
Upon
refeeding
these
two
microcosms
with
toluene
and
o-xylene
simultaneously
(100
,uM
each),
degra-
dation
of
both
compounds
began
without
a
lag.
In
the
microcosms
amended
with
toluene,
o-xylene,
m-xylene,
and
ethylbenzene
at
a
high
concentration
(total
concentration,
1.3
mM),
none
of
the
compounds
were
transformed.
In
microcosms
amended
with
benzene,
naphthalene,
and
phe-
nol,
only
phenol
was
degraded
after
80
days
of
incubation.
Neither
benzene
nor
naphthalene
was
transformed
after
more
than
300
days
of
incubation
in
microcosms
with
and
without
phenol.
No
significant
depletion
of
aromatic
com-
pounds
in
the
chemical
control
or
autoclaved
control
micro-
cosms
was
observed
over
the
course
of
300
days
of
incuba-
tion.
Enrichment
cultures.
Primary,
secondary,
and
subsequent
enrichment
cultures
retained
the
ability
to
degrade
toluene
and
o-xylene
to
methane
and
carbon
dioxide.
In
enrichment
cultures
that
were
always
fed
a
mixture
of
toluene
and
o-xylene,
both
compounds
were
simultaneously
degraded
with
no
evidence
of
competition.
However,
cultures
that
were
fed
toluene
only
for
more
than
2
years
could
no
longer
degrade
o-xylene,
and
vice-versa.
The
cultures
readily
de-
graded
toluene
and
o-xylene
but
did
not
degrade
m-
or
p-xylene.
Over
a
period
of
2
years,
the
rates
of
degradation
of
toluene
or
o-xylene
increased
from
5
,uM/day
to
50
jiM/day,
and
the
maximum
substrate
concentration
de-
graded
by
these
cultures
also
increased
from
about
200
jiM
to
2
mM.
The
optimum
pH
for
degradation
was
found
to
be
near
6.0
for
both
toluene
and
o-xylene,
consistent
with
the
fact
that
the
pH
of
water
in
the
Pensacola
aquifer
was
6
or
less
(15).
At
pH
7.0,
the
rate
of
toluene
degradation
was
75%
of
the
rate
at
pH
6.0,
and
at
pH
8.0
the
rate
of
toluene
degradation
was
40%
of
the
rate
at
pH
6.
A
decrease
in
temperature
from
35
to
20°C
caused
a
25%
reduction
in
the
rate
of
toluene
degradation.
Only
methanogenic
bacteria
are
known
to
produce
meth-
ane.
These
bacteria
exclusively
metabolize
simple
one-
or
two-carbon
compounds
and
hydrogen.
Therefore,
the
com-
plete
methanogenic
degradation
of
complex
organic
com-
pounds
such
as
toluene
is
believed
to
be
carried
out
by
the
cooperative
interaction
of
several
groups
of
bacteria.
Syn-
trophic
relationships,
such
as
interspecies
H2
transfer,
en-
able
reactions
that
would
otherwise
be
thermodynamically
unfavorable
(28).
We
attempted
to
isolate
pure
cultures
of
toluene-
or
o-xylene-degrading
organisms
from
the
mixed
cultures,
using
anaerobic
roll
tubes
as
described
by
Hungate
(19).
Colonies
were
obtained
after
3
to
4
weeks
of
incuba-
tion.
Some
colonies
were
transferred
to
liquid
medium,
and
slow
degradation
of
toluene
was
observed.
However,
the
slow
degradation
of
toluene
was
accompanied
by
methane
production,
indicating
that
the
colonies
were
not
pure
cul-
tures
but
a
mixture
of
fermentative
and methanogenic
bac-
teria.
We
did
not
succeed
in
separating
the
members
of
this
consortium,
perhaps
because
of
the
very
tight
syntrophic
relationships
that
probably
exist
between
the
various
organ-
isms
in
the
culture.
Theoretical
stoichiometry.
The
overall
theoretical
stoichio-
metric
and
energetic
equations
for
toluene
and
o-xylene
biodegradation
under
methanogenic
conditions
were
devel-
oped
following
the
method
described
by
McCarty
(26),
assuming
an
efficiency
of
energy
transfer
of
60%
(Table
1).
This
method
is
based
on
the
assumption
that
a
correlation
exists
between
the
free
energy
of
reaction
and
cell
yield.
On
TABLE
1.
Stoichiometry
and
energetics
of
toluene
and
o-xylene
oxidation
under
methanogenic
conditions
Type
of
Aromatic
Equation
equation
compound
Stoichiometrya
Toluene
C7H8
+
7.102
H20
+
0.072
NH4+--2.318
HCO3-
+
4.32
CH4
+
2.39
H+
+
0.072
C5H702N
o-Xylene
C8H10
+
7.788
H20
+
0.084
NH4+
-2.540
HC03-
+
5.04
CH4
+
2.624
H+
+
0.084
C5H702N
Energyb
Toluene
C7H8
+
7.5
H20--4.5
CH4
+
2.5
HC03-
+
2.5
H+
o-Xylene
C8H10
+
8.25
H2O--5.25
CH4
+
2.75
HC03-
+
2.75
H+
a
Includes
cell
(C5H702N)
formation.
b
The
data
for
computing
free
energy
changes
(AG"')
were
taken
from
the
studies
by
McCarty
(26)
and
Thauer
et
al.
(31).
For
toluene
and
o-xylene,
AGO'
were
-31.2
kcalmol
(ca.
-131
kJ/mol)
and
-40.3
kcal/mol
(ca.
-169
kJ/mol),
respectively.
For
comparison,
the
AG"'
values
for
the
aerobic
oxidation
of
toluene
and
xylene
are
-910
kcal/mol
(ca.
-3,810
kJ/mol)
and
-1,060
kcal/mol
(ca.
-4,435
kJ/mol),
respectively.
this
premise,
we
calculated
that,
theoretically,
4.32
mol
of
methane
and
2.32
mol
of
carbon
dioxide
would
be
generated
from
the
degradation
of
1
mol
of
toluene,
or
that
62, 33,
and
5%
of
the
carbon
from
toluene
would
be
converted
to
methane,
CO2
(or
HCO3-),
and
cell
material,
respectively.
With
these
equations,
theoretical
cell
yields
of
11
g
of
cells
(dry
weight)
per
mol
of
toluene
and
13
g
of
cells
per
mol
of
xylene
were
calculated.
The
amount
of
energy
available
for
microorganisms
from
the
oxidation
of
toluene
or
o-xylene
under
methanogenic
conditions
is
considerably
(about
17
times)
less
than
that
for
toluene
or
xylene
oxidation
under
aerobic
conditions
(Table
1).
Mass
balances.
Measured
methane
concentrations
were
consistently
between
85
and
100%
of
the
theoretical
methane
20
0
0,
ca
0
*.
rz
0
10
20
30
Time
(days)
40
50
FIG.
1.
'4C
label
distribution
in
mixed
methanogenic
cultures
fed
[ring-14C]toluene.
14C
in
the
volatile
(toluene),
nonvolatile
(biomass
and
nonvolatile
intermediates),
and
CO2
fractions
was
measured
directly.
"4C
in
methane
was
calculated,
by
using
theoretical
stoi-
chiometry,
from
the
mass
of
toluene
degraded
at
each
sampling
time.
dpm,
disintegrations
per
minute.
APPL.
ENvIRON.
MICROBIOL.

METHANOGENIC
DEGRADATION
OF
TOLUENE
AND
o-XYLENE
317
A
10
A_zoo
Toluene
*
data
_~
1000
-
model
6u
40
_
800
0
~~~10
20
Time
(days)
B_
o-Xylene
3.-
2u
@J
00-
0
0
400
\-moe
0
10
20
Time
(days)
FIG.
2.
Kinetics of
toluene
(A)
and
o-xylene
(B)
degradation.
Experimental
data
were
plotted
with
Monod
kinetic
model
predic-
tions.
The
biomass
yield
(Y)
and
the
initial
biomass
concentration
(X0)
were
measured
experimentally.
The
Monod
parameters
Ks
(half-saturation
constant)
and
ULm.
(maximum
specific
growth
rate)
were
approximated
by
nonlinear
regression
of
the
data
from
sub-
strate
depletion
curves
to
the
Monod
model.
The
Monod
model
parameters
for
toluene
were
as
follows:
Y
=
17
g
of
cells
per
mol
of
toluene,
XO
=
6.5
mg/liter,
Ks
=
30
,urn,
and
WUm.x
=
0.
11
day-'.
The
Monod
model
parameters
for
o-xylene
were
as
follows:
Y
=
17
g
of
cells
per
mol
of
o-xylene,
X0
=
8.4
mg/liter,
Ks
=
20
,um,
and
WUma,
=0.07
day-.
concentration
(based
on
equations
in
Table
1)
for
both
toluene
and
o-xylene
in
22
different
incubations
over
the
three
years
of
this
study.
Radiolabeled
toluene
and
o-xylene
were
used
to
confirm
the
formation
of
C02
(or
HCO3
).
The
14C
label
distribution
shown
in
Fig.
1
was
obtained
from
duplicate
cultures
fed
[ring-
14C]toluene.
The
radioactivity
in
the
volatile
fraction
(toluene),
the
nonvolatile
fraction,
and
the
C02
fraction
was
measured
over
time.
For
the
volatile
compounds
CO2
and
toluene,
the
total
mass
in
the
bottle
(aqueous
phase
and
headspace)
was
the
sum
of
the
counts
obtained
in
samples
from
the
liquid
phase
and
the
corre-
sponding
counts
in
the
headspace
determined
by
using
the
Henry's
Law
constants
for
C02
and
toluene.
It
was
difficult
to
measure
the
radioactivity
associated
with
methane
be-
cause
methane
partitions
almost
completely
into
the
head-
space
and
is
very
poorly
trapped
in
scintillation
fluid.
Since
0
60
450
o-
.-Xylene
0
600
1200
1800
24'00
Initial
Concentration
(jiM)
FIG.
3.
Rate
of
degradation
versus
initial
substrate
concentra-
tion
in
mixed
methanogenic
cultures
enriched
with
either
toluene
or
o-xylene.
we
found
that
near
stoichiometric
amounts
of
methane
were
produced
in
experiments
with
unlabeled
substrates,
we
calculated
the
theoretical
yield
of
radiolabeled
methane
from
the
measured
amount
of
labeled
toluene
degraded
at
each
time
point
and
plotted
these
calculated
[14C]methane
values
in
the
same
figure
(Fig.
1).
A
near
perfect
mass
balance
was
obtained
upon
summing
up
the
radioactivity
in
the
various
fractions
(measured
and
theoretical).
Experiments
with
methyl-labeled
toluene
or
methyl-labeled
o-xylene
also
yielded
near
stoichiometric
amounts
of
labeled
CO2.
Kinetics.
Over
a
period
of
2
years,
the
rate
of
degradation
in
enrichment
cultures
increased
10-fold
predominantly
as
a
result
of
the
increased
biomass
concentration.
From
protein
measurements,
we
estimated
the
cell
yield
(Y)
to
be
about
17
g
of
cells
per
mol
of
toluene
or
o-xylene
(standard
deviation
=
6.2,
n
=
8).
The
observed
yield
of
17
g/mol
is
marginally
greater
than
the
predicted
theoretical
yield
of
11
to
13
g/mol
and
may
indicate
that
the
actual
energy
transfer
efficiency
is
greater
than
60%
(assumed
in
theoretical
calculations)
and
might
be
closer
to
80%.
Anaerobic
systems
have
been
shown
to
have
higher
energy
transfer
efficiencies
(26).
Substrate
depletion
curves
for
toluene
and
o-xylene
were
obtained
for
a
range
of
initial
substrate
concentrations
and
for
a
given
initial
biomass
concentration.
The
initial
biomass
concentra-
tion
in
these
substrate
depletion
experiments
was
estimated
from
protein
measurements
and
was
assumed
to
be
the
active
biomass
concentration
(XO).
We
measured
an
initial
biomass
concentration
of
6.5
mg/liter
and
8.4
mg/liter
for
the
toluene
and
o-xylene
depletion
experiments,
respectively.
The
data
from
substrate
depletion
curves
for
toluene
and
o-xylene
were
fit
to
the
Monod
kinetic
model
(without
a
decay
term)
by
nonlinear
regression
(Fig.
2)
to
derive
values
for
the
half-saturation
constant
(Ks)
and
the
maximum
specific
growth
rate
(pm.x).
This
procedure
yielded
esti-
mates
for
K,
of
30
and
20
,uM
(+
30%)
and
for
IUmax
of
0.11
day-1
and
0.07
day-1
(+
20%)
for
toluene
and
o-xylene,
respectively.
The
doubling
times
for
the
stable
consortia
growing
on
toluene
or
o-xylene
were
therefore
about
6
and
10
days,
respectively.
Typically,
toluene
was
degraded
more
quickly
than
o-xylene,
consistent
with
these
kinetic
param-
eters.
Because
growth
was
slow,
the
rate
of
degradation
depended
strongly
on
the
initial
biomass
concentration.
At
initial
substrate
concentrations
higher
than
those
plotted
in
VOL.
60,
1994

A
300
Sterile
control
0
200
Im
irt
0
10
20
Time
(days)
B
1st
feeding
2nd
feeding
3rd
feeding
300
---
Sterile
control
__
U~~~~~~~~~~~~~~~
No
sulfate
200
-
56
mM
Msulate
-0---O
lOmM
sulfate
100\
0
0
5
10
15
20
Time
(days)
C400
300
200-
5.6
j.M
oxygen
1.6
jiM
oxygen
a~100-
No
oxygen
0
I
10
1
5
2'0
Time
(days)
FIG.
4.
Inhibition
of
toluene
degradation
by
alternate
electron
acceptors.
(A)
Nitrate
(NaNO3);
(B)
sulfate
(Na2SO4);
(C)
oxygen
(02)-
Data
are
the
averages
of
measured
toluene
concentration,
in
duplicate
vials.
Toluene
and
the
alternate
electron
acceptor
were
added
to
the
culture
vials
initially
(first
feeding).
For
the
second
and
third
feedings,
only
toluene
was
added
to
the
vials.
318

METHANOGENIC
DEGRADATION
OF
TOLUENE
AND
o-XYLENE
319
Fig.
2,
the
data
no
longer
fit
the
model
because
the
rates
of
degradation
were
considerably
lower
as
a
result
of
substrate
toxicity.
Substrate
toxicity
was
observed
at
an
initial
con-
centration
of
700
,uM
for
o-xylene,
but
not
until
the
concen-
tration
was
increased
above
1,800
,uM
for
toluene
(Fig.
3).
Effect
of
alternate
electron
acceptors
on
toluene
degrada-
tion.
The
addition
of
exogenous
electron
acceptors,
such
as
nitrate
or
sulfate,
slowed
down
or
completely
inhibited
the
degradation
of
toluene
depending
on
their
concentration
(Fig.
4A
and
4B).
Nitrate
was
more
inhibitory
than
sulfate
at
the
same
concentration.
Oxygen
was
extremely
toxic
to
this
consortium
(Fig.
4C),
significantly
inhibiting
degradation
at
a
concentration
of
5.6
,uM
(0.18
mg/liter)
and
completely
inhibiting
degradation
at
a
concentration
of
50
,uM
(1.6
mg/liter).
Survival
after
starvation,
pasteurization,
or
freezing.
Cul-
tures
were
able
to
survive
for
extended
periods
of
time
(at
least
1
year)
without
any
added
toluene.
Upon
refeeding
of
the
cultures,
toluene
degradation
was
initially
very
slow,
but
the
activity
was
recovered
and
the
rate
of
degradation
eventually
returned
to
prestarvation
levels.
Toluene-degrad-
ing
activity
was
lost
after
pasteurization
of
active
cultures.
However,
toluene-degrading
activity
was
maintained
after
pasteurization
of
cultures
that
had
been
starved
beforehand,
with
or
without
inoculation
with
methanogens
after
pasteur-
ization,
suggesting
that
all
the
members
of
the
community
degrading
toluene
(including
the
methanogens)
were
able
to
withstand
mild
heat
shock,
provided
that
the
culture
had
been
stressed
beforehand
to
induce
some
form
of
sporulation
or
cell
resistance.
Microscopic
observation
confirmed
the
presence
of
some
spore-forming
microorganisms
in
stressed
cultures.
Cultures
that
were
flash
frozen,
stored
at
-80°C,
and
later
revived
by
thawing
the
frozen
aliquot
in
sterile
anaerobic
medium
retained
the
ability
to
degrade
toluene
and
o-xylene.
Effect
of
alternate
substrates
on
toluene
degradation.
To
determine
whether
the
presence
of
carbon
sources
other
than
toluene
would
stimulate
or
inhibit
toluene
degradation,
a
variety
of
test
compounds
in
addition
to
toluene
were
fed
to
the
consortium.
Overall,
none
of
the
compounds
tested
stimulated
degradation
of
toluene.
Acetate,
hydrogen,
meth-
anol,
glucose,
propionate,
fatty
acids,
Casamino
Acids,
yeast
extract,
and
cysteine-HCl
were
immediately
used
by
the
consortium
as
growth
substrates
preferentially
over
toluene.
Only
when
the
test
compound
was
nearly
com-
pletely
degraded
did
the
degradation
of
toluene
begin.
These
inhibitory
effects
are
illustrated
for
acetate
(Fig.
SA)
and
glucose
(Fig.
SB).
Similar
trends
were
observed
for
the
other
compounds
tested,
which
were
immediately
used
as
sub-
strates.
Acetone
had
initially
no
effect
on
toluene
degrada-
tion
because
acetone
was
being
used
only
very
slowly
by
the
culture.
However,
after
all
the
toluene
and
acetone
from
the
first
feeding
had
been
consumed,
the
cultures
were
refed
both
acetone
and
toluene;
and
this
time,
acetone
degradation
began
immediately
(presumably
because
the
cultures
had
adapted
to
this
substrate)
and
toluene
degradation
was
inhibited
(Fig.
5C).
Low
concentrations
of
interspecies
me-
tabolites
such
as
H2
and
acetate
are
believed
to
drive
overall
degradative
reactions
in
methanogenic
systems
(28).
The
accessory
organic
compounds
tested
were
degraded
more
rapidly
than
toluene
and
preferentially
over
toluene
and
may
have
caused
transient
build-up
of
acetate
and
H2
to
concen-
trations
at
which
the
degradation
of
toluene
is
no
longer
energetically
favorable.
In
fact,
acetate
was
observed
to
build
up
during
the
degradation
of
glucose
and
propionate
to
concentrations
that
would
inhibit
toluene
metabolism
as
A
~~
lS0
k
9~~~5
mMac
100-
0-5
0
s
10
1S
20
25 30
Time
(days)
B
250
1.7mM
l
\17
mM
c
ucoses
100-
i
\.7
mM
glucose
n
50~~
170
lcc\
6
10No
glucoseg
0
5
10
15
20
25
Time
(days)
250
First
feeding
Second
feeding
I
I
~
*-0
_
Sterile
control
iso
m
acetone
w°
100
1
Mo
1
~~~2.5
m
aeone
6
0-50ImM
acetone
No
acetone
0
20
40
60
80
Time
(days)
FIG.
5.
Inhibition
of
toluene
degradation
by
alternate
electron
donors.
(A)
Acetate;
(B)
glucose;
(C)
acetone.
Error
bars
indicate
standard
deviations.
VOL.
60,
1994

320
EDWARDS
AND
GRBIC-GALIC
FIG.
6.
Scanning
electron
micrographs
of
methanogenic
cultures.
(A)
Toluene
degrading;
(B)
o-xylene
degrading.
Bar
=
1
,um.
shown
in
Fig.
5A.
Acetate
was
also
detected
as
a
transient
intermediate
during
toluene
degradation
(maximum
concen-
tration,
70
,uM)
and
would
accumulate
in
bromoethane-
sulfonate-inhibited
cultures
to
concentrations
up
to
300
to
400
,M
(bromoethanesulfonate
is
a
specific
inhibitor
of
methanogenesis
[16]).
Some
of
the
compounds
tested
were
toxic
at
certain
concentrations
and
were
not
metabolized.
This
was
the
case
for
cyclohexane,
carbon
tetrachloride,
and
benzene.
Cyclo-
hexane
and
carbon
tetrachloride
inhibited
toluene
degrada-
tion
at
concentrations
above
700
and
40
,uM,
respectively.
The
toxic
effect
of
some
substances
was
dependent
on
the
length
of
time
exposed
to
the
compound.
This
was
demon-
strated
by
amending
cultures
with
benzene
in
addition
to
their
normal
substrates
of
toluene
or
o-xylene
and
observing
the
rate
of
degradation
as
a
function
of
days
exposed
to
benzene.
The
rate
of
toluene
or
o-xylene
degradation
was
initially
unaffected
by
the
presence
of
benzene
(0.7
to
3
mM).
However,
the
rate
of
degradation
gradually
decreased
to
zero
over
a
period
of
50
to
100
days
of
incubation
in
the
presence
of
benzene.
The
rate
of
degradation
in
parallel
cultures
not
exposed
to
benzene
increased
over
the
same
time
period.
The
toxicity
of
the
substrates
toluene
and
o-xylene
was
also
dependent
on
the
length
of
time of
exposure
to
these
compounds.
We
observed
that
the
maxi-
mum
substrate
concentration
that
could
be
degraded
by
the
APPL.
ENvIRON.
MICROBIOL.

METHANOGENIC
DEGRADATION
OF
TOLUENE
AND
o-XYLENE
321
culture
depended
on
the
initial
cell
density.
At
higher
cell
densities,
higher
initial
toluene
or
o-xylene
concentrations
were
tolerated,
because
the
rate
of
depletion
of
the substrate
was
higher.
Faster
substrate
depletion
resulted
in
shorter
exposure
of
the
culture
to
high
concentrations
of
substrates.
At
a
low
cell
density
and
high
substrate
concentration,
cell
death
from
substrate
toxicity
was
faster
than
cell
growth.
Electron
microscopy.
Stable
consortia
growing
on
either
toluene
alone
or
o-xylene
alone
were
observed
by
scanning
electron
microscopy.
Rod-shaped
cells
predominated,
con-
firming
observations
by
light
and
epifluorescence
micros-
copy.
Two
different
types
of
rod-shaped
cells
(both
with
a
diameter
of
0.5
,um)
were
clearly
visible
in
the
culture
degrading
toluene
(Fig.
6A):
one
morphology
had
rounded
ends,
while
the
other
had
blunt
ends
abutting
the
next
cell
in
a
chain.
These
two
cell
morphologies
were
also
present
in
the
o-xylene-degrading
culture
(Fig.
6B),
although
the
round-ended
rods
appeared
fatter
and
more
ellipsoid
than
the
rods
of
similar
diameter
in
the
toluene-degrading
culture.
A
rod-shaped
cell
of
narrower
diameter
(0.3
,m)
was
much
more
prevalent
in
the
o-xylene-degrading
culture
than
in
the
toluene-degrading
culture.
A
notable
difference
between
the
micrographs
of
these
two
cultures
was
the
presence
of
a
large
web
of
exopolysaccharide-like
substance
in
the
o-xy-
lene-degrading
culture.
This
web
was
absent
in
samples
observed
from
the
toluene-degrading
culture.
The
blunt-
ended,
rod-shaped
cell
observed
in
both
micrographs
resem-
bles
the
obligate
acetate-utilizing
methanogen,
Methanothrix
soehngenii,
first
isolated
by
Zehnder
(12,
35).
DISCUSSION
Several
findings
from
this
study
have
important
implica-
tions
on
the
fate
of
monoaromatic
compounds
in
anaerobic
contaminated
sites.
Long
adaptation
periods
may
be
re-
quired
before
the
onset
of
detectable
levels
of
biodegrada-
tion.
Mutation
or
enzyme
induction
may
have
occurred
during
the
adaptation
period,
but
the
most
important
factor
contributing
to
the
long
adaptation
period
was
most
likely
the
very
small
number
of
active
microorganisms
in
the
sediment
initially
and
the
heterogeneity
in
the
distribution
of
active
microflora.
The
cultures
enriched
during
this
study
appeared
to
be
extremely
substrate
specific.
Although
monoaromatic
compounds
are
structurally
similar,
only
tol-
uene
and
o-xylene
were
degraded;
m-xylene,
p-xylene,
ethylbenzene,
benzene,
and
naphthalene
were
not
degraded.
The
addition
of
electron
acceptors
(such
as
nitrate
or
sulfate)
to
contaminated
anaerobic
sites
will
not
necessarily
acceler-
ate
degradation
if
the
indigenous
microbial
communities
are
acclimated
to
the
conditions
of
methanogenic
fermentation.
A
very
important
factor
influencing
the
anaerobic
biotrans-
formation
of
toluene
and
o-xylene
was
the
presence
of
other
organic
compounds,
either
natural
organic
compounds
or
other
components
of
pollutant
mixtures.
The
addition
of
preferential
microbial
electron
donors
(such
as
acetone,
methanol,
glucose,
fatty
acids,
and
amino
acids)
inhibited
toluene
degradation
in
our
experiments.
These
compounds
occur
naturally
or
are
frequent
cocontaminants,
and
their
presence
at
a
contaminated
site
may
prevent
the
degradation
of
compounds
that
are
more
difficult
to
degrade,
such
as
toluene
and
xylene.
Many
components
of
pollutant
mixtures
are
toxic
to
microorganisms
at
certain
concentrations.
The
degree
of
toxicity
is
dependent
on
the
substance
itself,
the
concentration,
and
on
the
length
of
time
that
the
microor-
ganisms
are
exposed
to
the
toxic
substance.
Microorganisms
have
evolved
defense
mechanisms
for
surviving
inhospitable
conditions
in
the
subsurface.
The
microorganisms
enriched
in
this
study
were
shown
to
withstand
starvation
and
heat.
These
same
defense
mechanisms
may
offer
these
organisms
some
protection
against
many
other
forms
of
stresses
present
in
natural
and
contaminated
environments.
ACKNOWLEDGMENTS
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.
We
thank
Fran
Thomas
(Biology
Department,
Stanford
Univer-
sity)
for
help
with
electron
microscopy
and
Ned
Black
and
Harry
Beller
for
critical
review
of
the
manuscript.
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