present,
two methanogenesis,
(Jorgensen,
believed
sulfate
can
important heterogeneous
particulate
many
1992).
can
studying
Introduction
behavior
anaerobic
marine production
potential
explored
and
from conditions
Sulfate
Abstraot
2
3
1
University Université
Doane
recirculate
anaerobic
regulate,
to
of
the
NO
Two
ion
The
environment
College,
reducers
these
to
as
establish
could
the
role
same
with
for
organic
and
BUG
Pierre
of
as dominate
specific
organic
1980) in
study
primary
in
Washington,
metabolic
under
metabolic
constraint metabolic
syntrophism
of
potential
terminal
back
many
assembly
Crete,
cocultures
marine
reflect
environments.
archaea
IS
et
converting
and
Barbara
matter
of
Marie
and
AN
syntrophic
into
to
NE,
metabolic
matter.
yielded
pure sulfate-free
of
in
a
each
methanogenesis
ISLAND:
physical
Microbial
Curie,
systems electron
environments,
utilize anaerobic
Seattle,
capabilities
the
USA.
the
potential
mineralization,
and,
capabilities
of
of
cultures
Clement’,
among
complex
aerobic
cocultures,
group.
organisms
results
Paris
Particularly
determine
locally
Some
USA.
CO 2
pathways
relationships
should
and
acceptor
VI
THE
conditions,
Diversity
niche
environments.
local
are
in
as
Nonetheless,
are
regions
chemical suggestive
sulfate
France.
organic
COCULTURES
obtained
of
Michelle
the
terminal
dominant
be
associated
sulfate
MESSY
is
even
only
related
for
the
an
salt-water
for
laboratory
thermodynamically
for
in
minimal.
2002,
sulfate
supporting
reducers
transformation,
material
the
in
with
one
parameters
the
the
and
SRB
Graco 2
electron
reducing
of
sulfate-abundant
in
BUSINESS
terminal
remineralization
In
both
Woods
with
of
organic
syntrophism.
reducers
case
hydrogen-consuming
diverse
a
and
sulfate
When enrichments
constitutes
have
the
to
complex
groups
and
the
the
acceptor.
and
simple
fresh-water
of
Hole,
from
tested
bacteria
anaerobic
the
activity
life
matter
Nicolás
reduction,
sulfate
with
substrates.
the
fluxes
anaerobic OF
unbalanced;
ability can
the
(Gottschalk
MA
compounds
food
SRB
This
a
environments.
interaction
WORKING
(Fig.
consequential
and
of
environment.
mineralization,
very
of
generally
(SRB)
is
Pinel 3
microorganisms
and
organic
sulfate
other
to
observed
web
enrichments
unavailable
bacteria
a
1).
powerful
Despite
act
environments,
hydrogenotrophic
accumulation
In organisms.
when
organisms
are
&
carry
and
as
nature,
reducers
between
be
material
Peinemann,
WITH
utilize
fermenters,
syntrophic
gases
dominant
However,
effects
sulfate
recovered
out
tool
and
in
from
these
under
that
that
the
the
are
was
The
the
the
for
in
of
is
on
a a
used
syntrophic
energy reaction
SRB,
fermentative
such acceptor,
depending
substrates, reflect
SRBs systems
be
1988).
Fig.
freshwaters
system,
recovered
to
1
as
the
Anaerobic
as
the
Although
(Table
support
Despite
Consequently,
(H 2 )
methanogens.
coexist
a
and
methanogenesis
energetics
(mutual
specific
on
from
group
mode
can
from
systems
are
1).
the
the
the
metabolic
under
H 2
a
be
In
able
has
feeding)
available
of geochemical
enrichment
single
growth
metabolic
to
used
this
of
energy
and
been
In
the
anaerobic
to
low
the
way,
pathways
by
the
establish
soils
should
of
relationship.
SRBs molecular
reported
reaction
substrate
conservation.
another
the differences
absence
cultures
the
methanogenesis
and
conditions,
partner
be
may
products
syntrophic
redox
the
may
organism,
as
weight
and
of
from
reaction
shift
being
dominant
between
organism,
sulfate,
attributes
be
the
2
of
the
although
from
made
fatty
very
one
relationships
presence
appear
generating
same
may
SRBs SRBs
type
acids
anaerobic
and
of
versatile
more
not
source
the
the
as
the
of
and
or
can
to
be
a
environment.
favorable
activity
a
organism’s
dominating
absence
two
aromatic
with
energetically
methanogens,
more
material.
shift
with
respiration
(Winfrey
organisms
H-consuming
negative
from
respect
of
of
if
compounds
one
These
processes.
metabolism
the
a
The
a
&
suitable
favorable
or
respiratory
to
to
both
Zeikus,
can
product
change
metabolism
two
the
utilization
fermentation
establish
groups
organisms
other
metabolic
(Widdel,
electron
can
1979).
in
of
to
free
to
will
the the
can
be
of
of
a
a In
producers
conditions
The
methanogen
goal
Methanogenesis
Table
Fiebis
methanogens.
and
natural
syntrophic
microorganisms
several
However
complex
less
which
substrate
which
Fermentation
relationship
Syntrophic
of
hypothesis
the
important
1.
Based
this
and
One
both
the
anaerobic
methanogens
Changes
The
metabolic
and
substrate
or
research
Gottschalk,
and
mutual
relationship
in
of
term
partners
any
on
SRBs
establish
the
than
locally
is
anaerobic
the
of
other
depend
environments
“syntrophism”
Gibbs
types
completely
that
syntrophic dependence
is
Glutamate
Lactate
Ethanol
H 2 /C0 2
reported
Glutamate
Lactate
Ethanol
have
for
utilize
depend
to
type
1983;
recovered
locally
is
syntrophic
anaerobic
explore
free
participating
been to
probably
of
environments,
the
Phelps
energies
a
metabolic
entirely
nutrient.
systems
to
shown
great
on
H 2 exploiting
recovered
the
is
CO 2
SRBs
organisms.
each
for
20
used
CH 3 COO
+ 2
+
2
4 +24
20
2
4H 7
relationships 2
et
potential
one
under
HCO CH 3 CHOHCOO
H 2 +
CH 3 CH 2 OH
CH extend
CH 3 CHOHCOO
CH 3 CH,OH
and
on
to
a!,
Glu
Glu
in
the
demonstrated
The
versatility
+
from
CH 3 COJ
other
to
function
way
+
4 each
CO 2
a
1985).
water
+
reduction
minimal
+
3 standard
H 2
+
Equations
H 2 0
SRBs
denote
complex
syntrophic
24
+2
the
24
on
marine
for
of
H 2 0
cannot
The
other
butyrate
+
H 2 0
without
+2
the
+
syntrophism
life
HC0 3 as
catabolic
with
should
of
those
8
+
and
H,O energy
CH 4
aerobic
+
butyrate
net
of
=
+4
8W
to
and
cooperation
feeding
in
sulfate
that
H 2 0
be
24
+
relationship
syntrophic
CO 2
+
perform
H,O
methanogens
H 2
the
the
CH 4
cooperations
freshwater
2
overcome
NH 4
have
=
the
H 2 0
yields
2CH 3 C00
producers
=
+4
bacteria
20
to
laboratory cooperation
=2
reducing +
2CH 3 C00
2CH 3 COJ
and
network
between
NH 4
methane
H 2 organisms
+
CH 3 CQQ
the
a
conditions.
5
(Mclnemey
of
metabolic
in
CO 2
anabolic
+
by
systems.
can
ability
+
20
other
(during
aerobic
between
bacteria
in
2H
(Schink,
+
is
+
simply
a
(Bryant
+ CO,
of
+
frequently
H
pure H
that
sulfate-depleted
use
6
+
2
other
organisms
to
activity
-190.0
-134.97
-131.0
fermentation)
59
et
.397
+
4214
organisms
(Kj/mol)
activities
to
LIG 0 ’ of
culture
adding
(SRB),
organisms
act
9.6 et
a!.,
1997).
organisms.
SRBs survive
al,
as
degrade
1979).
and
1977;
of
a
the
H 2
with
The
and
a co
are
in
of
in in
Modified Inoculations
atmosphere. sterile followed
added Medium
and was
additions,
Media:
SRB
Syntrophlcoculture
were
material,
enrichments
obtained
syntroph
SRBs, a determine
The
the
isolate, could
Rationale organism The
organisms) respiration
rather we
manner by favorable
metabolic between
microbial
organism
an
Methodology
known
medium
adding
removing artificial
cooled, did
basal
not sulfate-reducing methanogen
media to
only
having
than but
was not
basal
by
Coculture
SRBs.
lowers
achieve from
gassed methanogen, partners
a
SRBs
of
whether
for
fresh
for
partners activity,
in were
1 since
does gassing
without grow
were
add Tubes
freshwater (absence
modified
and was
medium known respire
ml
selection
medium
been the
the
artificial
material
water
and
the sulfate
made
with
of not
its
additions
a
pure
used
dispensed
when
SRB,
medium: H 2
is were
were locally-derived
each
enriched with final
and hydrogen
Se-Wo
partial
growth
methanogens
metabolic
generate the
of
anaerobically,
bacteria
N2/C0 2 ,
of
for
medium
for using
culture
with
of
and
and ion environment allowing sulfate) the
media.
from
suitable capped
provide
growth
some
sterile
obtained
concentration
cultures:
the
syntroph
pressure
made to utilizing methanogen
is
solution.
2) the
into
sterile
in
coculture production.
Sippewissett
hydrogen,
not
(SRB)
the
(Methanococcus kind.
was they
stoppered
capability,
classroom
(filtered)
knowledge
with should
of
products
something it
Syntrophy
sterile
related
may
from
media
organisms
syringes. 2
culture: from
to
prepared
were
using of
it
or
sterile
In
enrichments
that
continue From
not be of
for
the
of more
filter-sterilized
contrast,
Baich
makes
and
N 2 /C0 2 in
to
three
and
both
a
established
of activities
and 4
directly
readily
10 making
that sulfate
marsh
gas
Hungate order
sulfate for
ready
0.5
is
in
syntrophy
could
SRB organisms
autoclaved.
mM.
SRB
tube
an with
inaripaludis) it the
and
500
they a locally-derived
M
pure
possible
to
special
by
obligate
(marine)
selected
affect source concentration,
demonstrates activity
as
other
methane.
establish
sterile
in
ml makes
its and “force”
in
rubber most
All
the
when
culture
the
the
batches metabolic
an
is
stocks
in
methanogens
its
organism.
Hungate additions
not
case
likely
of
After for
previous
stock
anaerobic the
electron
the
hydrogenotroph
for
were
stoppers
the
growth
and the
was syntrophic
both
the
possible
is
Removal same
reaction
the
as
without
of
SRB
enrichments;
SRBs autoclaving,
the
contained one
solutions,
present).
methanogensis.
the
selected
SRB
activity.
technique.
per
marine
coculture
study
or
to
1-2
physical
In
growth
acceptor.
and produces
from
hood conditions
metabolic
by
to
the
the
(between
this to
substrate weeks.
more
relationships
of crimped
omitting ferment
were
continue
because
and
medium
organisms
recipe
Ceder with
In substrate
the case,
It
(and
of
the
space,
in
energetically
Five
our
is
H 2
one used
freshwater
H 2
two
present
Anaerobic
The which
N2/C0 2
these
medium
activity.
syntrophy
a
therefore
or
closed. substrate
cultures,
Swamp
below.
sulfate to
from it
marine
ml
specific
These
usually
were
other
in were
was
SRB 1)
is with
that grow
two
of this
to an
the
in its
were
syringes.
were
rnarzaludis,
The
incubated Hungate
dispensed,
Cell
and
Culture
lyophilized medium
aseptically Methanogen
morphology
shaped
was
cultures
was
air of
SRB
following:
Source
Sulfate-reducing
Headspace
10
bubble
crimped
potential
growth
added
prepared
inoculated
noted
media
mlvi.
organisms
finalpH7.2—7.3
conditions:
30
1 30
1
1
cultures:
11
final
were
1 30
1
1
1
11
with
stoppers
Care
mMNa 2 S
ml
on
ml
mMNa 2 S
ml
ml
ml
amendments
was
mM
mM
basal
to
into
culture
mM
basal
was
(or
gas
Media were
closed.
and
an
varied
SL
7
cultures
pH
Se-Wo
SL
syntrophic 7
the
notable
was
a
in
vitamin
present
vitamin
NaHCO 3
enclosed
Na 2 SO 4
was
NaHCO 3
and
cells
22
headspace
10
carried
marine
one
7.2
10
fresh
bottle
a
supplemented
were
bacteria
and
taken
added
was
N2/C0 2
(trace
ml
(as
(trace
N 2 /C0 2 .
—
maintained
solution
of
Incubation
for
were
apparent
7.3
with
water
solution
after
predominant
Baich
solution
prepared
would
aseptically,
the
made
out
mineral
partnerships
not
shaker,
a
aseptically,
mineral
mineral
were
distinct
SRB
gas
in grown
anaerobic
atmosphere,
inoculation.
to
mineral
where
tubes
Baich
be
introduce
under
of with
medium
was
with
in
enrichments
grown
in
solution)
expected
solution)
odor
80%
in
strictly
and
50
under shapes.
in
necessary
tubes
medium
ethanol
and
the
were
a
gas
ml
microscopic
a
the
of
in
medium
H 2 /20%
air
temperature
stationary
and
the
Incubation
Pfennig
sulfide
containing
in
anaerobic
enrichment
composed
bottle
anaerobic
in
5
when
These
tube
or
in
inoculated
the
enrichment
(e.g.
acetate
a
CO 2 .
“topped
comprised
headspace.
stoppered
suitable,
taking
upon
bottles.
incubator
cells
observation).
addition
was
regulated
conditions
conditions
5
of
as
cultures
opening
ml
Five
were
by
samples.
the
in
substrate,
off”
cultures),
sulfate-free,
Source
of
the
with
injection
methanogen,
of
at
of
ml
actively
sterile
Medium
with
to
30°C.
dark
basal
with
the
inhibitor).
in
a
(80%
of
30°C.
material
sterile
Hungate
sterile
Successful
to
cap.
short
medium, media
at
media
of
motile.
fresh
a
30°C
medium.
H 2 /20%
was
final
cells
Although
Hungate
rods
Methanococcus
media
for
was
Cultures
water
prepared
containing
tubes,
until
concentration
capped
with
enrichments
and
enrichment
C0 2 ),
dispensed
so
Cultures
turbidity
stopper,
mineral
cellular
which
sterile
vibrio
that
were
with
and
the
the no
All
work
column.
Gas
phase.
Acetate
Nova-Pak
330
derivatized
Glutamate
phthaldialdehyde!ethanol/2-mercaptoethanol/borate
90:10
disappearance
Glutamate
Agilent
HPLC.
Samples
sterile As
were
S
At
examined
numbers
produced
Turbidity:
counted
amounts Cultures
wavy
numbers
buffer.
chamber.
analysis), withdrawn
aliquot
Growth
Cell
(slight),
samples
or
Chromatoraphv intervals
described
for
compared
numbers:
360
morphology
Standards
mobile
1.5
was
1100
lactate,
Bio-rad
was
were
of
in
to
of
or
were
15
C18
or
tim.
for
eluted
a
and
were When
glutamate
ml
utilization
cultured
all
from
culture
measured enrichments
+
approximate
final
series
during
centrifuged
il
filtered
turbidity,
above,
treatments
(unequivocal
column
noted
microcentrifuge
the
phase of
similar
with
Aminex
analyzed
of
of
at
the
possible
inoculum
as
HPLC
cell
the
that
1
approximately
this
cells
the
uninoculated
(GC):
culture
in
just being
and
aliquots
were
directly
consisting
was
(standard
sensitivity
pellet
produced
which the
experiment
substrate.
HP7O
cell
were
and (10,000 was
with
before
10mM
with
to
cell
prepared
different
cell
growth).
measured
tube
distinguish
of
controls.
suspension
resuspended
determined
of quantitated
a
was
column.
autosampler.
numbers,
from
tubes,
count
Shimadzu
approximately
analyzing
acetate
reversed-phase
cell-free
as with
inoculum
rpm
media
of
2
used
for
from
(
the
for minutes.
The
(Methanospirillum
methanol:acetate
each
a
x
and
For
by
acetate,
were
SRB
culture
as
1
standardizing
as
5
was
in
at
the
as
GC-8A ml
in
on
culture
6
minutes),
organic
a
analyzing
of
stored
a
3
glutamate
0,
to
order
negative Desulfovibrio
prepared
from (filtered)
the
confirmation
tuberculin
days
approximately
counted
5,
Standard
5-12
cell
similar
column).
fluid,
and
HPLC.
gas
medium
buffer methanogens
frozen
to
acids
numbers,
during
x
the
12
chromatograph.
control.
use
using
and
samples
retention
the
2%
using
was
106
days.
syringe
buffer/methanol/tetrahydrofuran.
concentrations
All
and
was
supematant
(OPD),
until
Detection
used
machine.
formaldehyde
approximately
of
cells).
11
were
cells/ml.
samples
0.0008
alcohols,
1:2
first
and
readily
a
cell
days),
100—200
Turbidity
for
time.
of
by Petroff
analysis
and
methanogen
prepared
inoculations
and
growth.
standardization
derivatized
cell
culture
N
a
of
were
identified
Initial
removed
tubes
The
and
sterile
H 2 S0 4
analyzing
morphology,
of
glutamate
Hausser
was
was
tl
in
analyzed
sugars.
column
equivalent
1
and
were
Culture
of
medium
1X
inocula
and
needle.
to
scored
made
conducted.
as
culture
by
(for
placed
phosphate
SRB,
with
10
counting
mobile
visually
with
was
Might
its
was
of
on
HPLC
tubes
using
mM
as
these
were
the
long
This
for
cell
and
was
an
o
at
in
0,
a a
presence
not UV to
Microscopy: Methanogens
Methanogens
Control
Control
Control Control
Exp
Exp Exp
Table AT-bOO
Analysis Shimadzu
were
approximately
operated
For
analyzed
UV
autofluoresce.
illumination
analysis
prepared
2
light.
General
of
of
on by
at GC-8A
the
bacterial
80/1000
It
are
an
utilizing of
methanogens.
can
and
0.25
is
Experimental
and
archaea,
bacterial +
+
possible +
-
3 2
oven
SRB
1
at
This
used
be
minutes.
observe
an
acetate
Chromosorb
oven
the
technique to
distinguished
temperature
and
to
generate
methane
Design
gas
formation
only
temperature
first
Standards
contain
chromatograph
the observe
W-AW
was
+
a
+
+
+
-
+
+
Methanogen
production standard methanogens.
a of
under
utilized
from
molecule,
of
of
75°C.
(Alitech).
7
a
1,
175°C
field
culture 10,
Eubacteria
curve.
for
under
with
100,
under
F420,
Methane
with
examination
conditions
Eubacteria
+ -
+
+ +
+
+
and
Substrate
a
culture
the
phase
that
Porapak
10000
by
following
autofluoresces
eluted
microscopy,
fluorescence
conditions,
was
do
of
ppm N
not cultures
measured
from
packed
80/100
methane
have - + - - - -
- Inhibitor
the
then
when
samples
to
F420
column:
microscopy.
column, by
(in
ensure
column
switch
using
exposed
N 2 /C0 2 ) and
were
10%
the
do
and
the
to in
enrichments
enrichments by
for
maripaludis.
vibrioid
Fig.
Methanococcus
(SRB1 or
produced
Results
detection
methanogens.
+
ethanol
2.
O.OOE±OO
Methane
5.OOE-04
I
Methanogenic
2.OOE-03
1.50E-03
2.50E-03
.OOE-03
or
Methane
and
similar
SRB2)
utilized
as
of
alone,
Their
rod-shaped -i--——’ - - -
-
production
substrate
autofluorescent
maripaludis
Autofluorescent
production
was
amounts
methanogenic
or did
—______
cultures
used
in
not
only
as
cocultures
cells,
contain
related
of
as
was
when
and
on
methane.
a
cells
source
the
all
character
the
cells
observed
to
considerable
either
three
treatment
at
characteristic
with
SRB
a
were
of
Methane
wavelength
syntrophic
experimental
of
BES
enrichments
was
8
after
not
the
conditions
Treatments
confirmed
numbers
added
detected
twelve
two
was
cocci
partners.
of
Sippewisset
detected
(Fig.
substrates
(Fig.
420
days
of
in
morphotype
with
native
nm
cultures
2).
3),
in
in
CM. DCeder
•
•Sippewissett
epifluorescent
(Fig.
most
All
Sippewissett
indicating
the
contained,
SRB
methanogens.
nmripaludis
cocultures
Swamp
inoculated
cocultures
3),
of
of
enrichment
the (Acetate)
Col
a
(Ethanol)
Methanococcus
diagnostic
(Ethanol)
that
cocultures
in
microscopy
on
addition
with
with
the
glutamate
cultures
SRB
SRB
lactate
trait
with to
negative
methanogenic
from
Fig.
10
days
3 SRB2
—
of
control
Phase
incubation.
(a,b)
cultures.
contrast
shown).
and
SRBI
Methanogenic
(left
Negative
column)
(c,d)
controls
methanogenic
and
cells
epifluorescent
(e,f)
detected
did
9
cocultures
by
not
blue
present (420
with
autofluorescence,
nm;
any
Methanococcus
right
autofluorescent
column)
and
microphotographs
inaripaludis
present
cells
only
(typical
after on
was
enrichments
corresponding
result
day to
was
maripaludis
comparable
SRB2 removal)
methanogen
the
detected
five
observed
Growth cell
considering
Fig.
or
Glutamate
z
E
D
E
glutamate
was
0.00
5.008+07
1.008+08
numbers
1.500+08 2.008+08
2.500*08
3.00+08
4
SRB3, 3.50E18
4.DOE+08
(Fig.
in
8÷00
Change
number
in
component
for
lactate
on
cultures
inhibited
I
these
1
5).
the
lactate
suggesting
the
supported
for
had
in
No
of
cultures
coculture
experiments.
Cell
continued
——S3-’SRB+Meth
—O--M.
——S2-’SRB”+Meth
—*—S3-”SRB”
—*—
—X—S3-’SRB”*Meth*Inh —l—-S2-’SRB’*Meth+Inh
—O--S2-’SRB’
—a--
(Fig.
SRB
growth,
reached
through
cells
tO
was
had S
Sl-SRB’*Meth+Inh
Si-SR
Numbers
1-S
mailpaludis
1
4).
R
growth
alone, R’+Meth
B’
with
detectable
a were
been
of
as
minor
undetectable
the
increase
SRB
SRB3. determined
through
In
obtained
or
excluded,
for
addition
contrast,
for
1
effect
all
in
and
in
the
10
time
cultures
the
Days
cell
t5
M
with
coculture
concentrations
of
of
by
or
on
cultures,
a
maripaludis
10
the
numbers
nearly
a
where
glutamate
glutamate
considerable
containing
mM
hydrogen
plus
five-fold
including
methanogenesis
BES
from
BES
in
on
for
to
either
most
scavenging
day
glutamate
increase
tb
on
the
all
increase
those
the
5
treatments
cocultures.
cultures,
the
until
same
controls
in
SRB1
(hence
when
in
cell
activity
day
substrate.
cell
a
numbers,
containing
or
surprising
compared
10
Similarly,
where
hydrogen
numbers
SRB2
on
of
the
By
the M
with
whereas
six
and
10.00
12.00
14.00
16.00
18.00
0.00—
2.00
inhibitor
4.00
8.00
6D0
Fig.
Growth
fourteen
-]
Fig.
I
the
0
6
4.0(IEl07
6.{HIE+07
8.
N
I
I.20E+O5
I 2
I
5
Fractional
OCeder
•
methanogenic •Sippewissett .OIIE*08
.411E+{IIt
.tiI)E+(B
(10
()OE+O
Sippewissett
respectively
E+(17
Change
on
times
J
1
-
Swamp
lactate
—*—S3-”SRB”+MeIhlinh
——S2-”SRW+MeIh
—0—- ——Sl-SRB”+MeIh+tnh
—•—
—A-—
—+--S2-”SRB”
-X--S3-’S
(Acetate)
Col
*S3’SRB”-’MeIh
in
(Ethanol)
Changes
higher
(Ethanol)
M.
S2-SR.W*MeIh-inh SI-SR
SI-
Cell
Fractional
rnanpsludis
S
RB’
RB
(Fig.
ElM
for
coculture
Numbers
(cell
cell
Oh
in
Cell
SRB2
6)
#
Cell
Control
coculture/cell
counts
Number
C>
through
Numbers
on
Treatment
cultures
Change
lactate
than
11
#
control)
time
the in
Days
in
was
5
cocultures
containing
Cocultures
on
culture
Lactate
unaffected
S
4
with
SRB1
SRB
by
and
1
active
+
A
alone
M
maripaludis
methanogenesis,
or
the
coculture had
5
glutamate
stoichiometric as
independent thermodynamics
within
Discussion
technical
The
numbers
(Fig. methanogens
substrate. amount
functional
Glu
-
U
©
well.
5.OOE+07
I
2OOE+08
I50E+08
2
consumption
OOE+08
+
50E+08
6).
The
the
6
Fig.
Insignificant
H20
difficulties.
According
of
-
fermented
- showed
Similarly,
individual
rationale
from methanogenesis,
7
change
—O---M.
—+— ——SI-SRB+Meth+Inh
—.--—
—
Changes
relationship
+2
*
were
S3-SRB+Meth
SJ-SRB+Meth+Inh
S2-SRB+Meth
S2-SRB+Meth+Inh
S
of
I-
hydrogen
H
maj-ipaludis
a
patterns
SRB
nearly
10
(Eq. absent
the in
to
for
growth
in
->
limited
reactions
cell
Cell
the
combined
1)
understanding
between
sixteen-fold
5
or
for
scavenging
numbers
NH 4
general Numbers
when
was
inhibited
growth
substrates
by
+
present
hydrogen
metabolic
ethanol
5
virtue
was
equation through
CO 2
increase
was
by
can
syntrophic
Days
t5
other
observed
BES.
on
12
+6
be
present
was
of
and
time
cultures
explained pathways,
CH 3 COO
when
than
product
for
However,
glutamate provided
on
relationships
in
the
in
Ethanol
glutamate
compared
cultures
with
ethanol
removal.
fermentation
with
+2
overcoming
with
as
is
SRB3,
butyrate
0.2
a
110
the
to
were
with
active
cultures
thermodynamic
lies
mol
the
energy
Growth
either
in
SRB2
not
control
+
H 2 methanogenesis,
of
energy
the
H 2
with
per
explored
source.
glutamate,
with
on
advantageous and
cultures
each
(Eq.
SRB1
limitations
glutamate
argument
or
ethanol
Minimal
mol
1).
due
without
when
the
of
cell
to as
glutamate
which
our
has
facilitating
amount
above-mentioned
those
removal
SRB3.
substrate
hydrogen
illustrated
glutamate,
The
=
methods.
been
-100.0
150.0
100.0
limited
-50.0
cultures
50.0
translates
The
0.0
Assuming
The
Fig.
Thus,
of
could inhibited,
-
1
level
as
partial
role
in
8
methane
observed
free the
-8
Free
substrate
influence .—..
‘
Fig.
in
thus
in
hydrogen
of
into
—
—--—Acetate
phosphorylation.
energy
question
pressure
Energy
our
such
SRB
—
8,
methanogenesis
should -7
-‘-S account
such
produced
where
Butyrate
Glutamate
Lactate
increase
Ethanol
experimental
suggests
an
of
remains
enrichments.
Dependence
a
concentration
-6
under
from
the
explanation
yield
small
Free
for
the
hydrogen
in
in
the
free
which
Energy
otherwise
a
amounts
-5
within
effect
cell
the
Glutamate
syntrophic
observed
in
Log
set
on energy
numbers
Lower
the
is
glutamate
Dependence
methanogens
H2
112
up,
-4
on
the
would
concentration
correct,
of
Partial
Partial
degradation
standard
growth 13
I
even
from
usable
hydrogen
growth
relationship
-3 amounts
.5—. fermentation
%
presumably
Pressure
for
Pressure
the
after
then
methanogenic
on
.5-
numbers
range
conditions. SRB1
-S
H2
in
are
-2
.5-S reaction
‘•5.
(atm)
stoichiometrically
measuring
complete
on
of
cultures
Partial
than
absent,
for
glutamate
methanogenic
the between
5-
that
remain
-l
independent
energy
is
expected
Pressure
fermentation
it
containing
plotted
or
cocultures
fermentation
can
the
0
below
where
the
in
conservation
not hydrogen
as
such
hydrogenotrophic
would
equivalent
cocultures
1
be
inhibitory
methanogenesis
from
a
either
of
involving
function detected
-S.
cultures,
of glutamate
5.
suggest
content
2
hydrogen
SRB2
the
through
to
using
levels.
of
with
initial
but
the
the
or
in the
a is
to
based
or resembles,
system One
propose specially
growing production favorable,
time Nonetheless,
disappearance by
suggests compared methanogenic
the
of
for determine
subsequent measured
jimol.
organisms we
62.5 With
(50 glutamate, 12.5 of hydrogen
reveal the
acetate
can relationship
1997), methanogen
establish
glutamate.
cultures
maintaining methane
longer way
and were jimol
intermediates
effect
be
on
jimol;
[Lmol;
From
further
The
until
This
Growth
the
that
proposed the
ethanol
produced
technical
that
considering media.
to
in
to periods total
which and
levels
production
working syntrophic substrate
effect on
of methane two
such
quantity
capable or
the c)
consumption can the of
dynamics
determine
A hydrogen
the however
fermentation
any
lactate
and
cocultures
methane. d)
If
and strongly
glutamate)
observed
on
syntrophic
fermentation
thermodynamically observed be
latter
active
However,
as further
corresponding of of
112.5
that
of
difficulties.
of from claim
methane as
calculated.
lactate
that
the
a methane
with production is serving
of
growth
the
time
containing
well
this
relationships substrates
through
if
does
close
fermentative
removal
p.mol
the
cases
the
methanogenesis
performing
suggested
not
dependent degradation
methane
Since results
of
on
growth
to of
syntrophy
enrichments
of
would
as
relationship
cultures
initial
this status
not
production,
in as to
on lactate
ensure syntrophism
can
if
the the
in
listed
the
The
the
magnitude,
the
that
preceded
fermentation by
involve stops.
we
lactate
methane-negative our
can
SRB1
does
thus
butyrate
was
hydrogen
result final
production
fermentation
of
by fermentation
base
the following
with above
can
expected cessation
with on
favorable
with be
these methanogenic is
of
organism
the
be
equation
sustained
not
methanogen
based
products,
hint
between
cocultures
hydrogen
suspect rather fermented
acetate for
in the
measurements
hydrogenotrophic
suspected.
by
SRB1
on
metabolic
generated,
is
should
SRB2 that
approximately
14
exclude
functions
the
at
facilitated
produced
syntrophic
taking
cases
from
on
of
observed
of
the
incipient
conditions
observed
than
syntrophic
1, were
of
that present showed
reactions by
methanogenesis.
the
organisms
lactate
i.e.
does
starting
be
partial glutamate.
by
a for
controls growth are
place,
carry-over is
glutamate
organisms
an degradation simple
interactions with
the
acetate, involved, allowing
syntrophic cannot
accompanied
possible: fermentation
SRB (case
on
that fermentation
not syntrophic
incipient
an
is
for
total
in
pressure in
10
the with
for
relationship.
the
pure
organisms
minimally
1 increase
of in the were (see
fermentation
seem
the
question,
a.). imol
As
butyrate,
with
cultures
amount
be
the
cultures
coculture substrate
SRB in
greater
methane
our
same
cocultures
a)
of
cultures,
results).
has systems.
Nonetheless, would
SRB1 SRB1
unavailable
dismissed
syntrophic
Changes
fermentation A
relationships.
to
H 2 ,
active
either
experimental
(see
1
of
by
of
in
been
simple
cocultures
and
of
be
for
should
the
growth,
glutamate.
thermodynamically or
and hovered
butyrate. From
cell
have
from data
levels without be
CH4
background),
enrichments.
of
the
This
2.5
the methanogenesis
affected M.
Measurements
reported
the expected
in
methane,
numbers
on
glutamate
fermentation to a
the butyrate the
be
maripaludis relationship, the imol
on degradation
to
cell would
would presence presumably observation
in follow
priori. around
on of
glutamate,
monitored
potential Given
us
inhibitor.
Similar
theoretical
conditions
previous
substrate
Instead,
numbers order
lactate by
lactate.
(Schink,
CH 4 ;
due
amount
rise
when
would
reach
or
with the
the we The
that
This
2.1
to
the
to
to
of
b)
to
of
of it
Mclnemey
Nature,
syntrophic
Jorgensen
identification,
al.(Eds). Gottschalk
Fiebig
sp. Appi.
Bryant
lactate
Bibliography
hitherto
they
Although
entirely corresponding
relationship
our
water
as
considerable methanogen
conditions.
environment
failed fresh
fermentative
such sulfate
environments.
consideration.
considering
maripaludis
is
fermentation
of
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fermenters
most
ethanol,
source
provide
Environ.
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or
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water
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&
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likely
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our
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of
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should
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&
(fresh),
(1982)
amounts
organism the
with
of
Gottschalk
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syntrophs,
starting
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media
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experiments
due
S.
lactate
cambell
potential
limiting,
this
interactions,
interests
patterns
any
Peinemann
due
unlikely
hydrogenotrophic
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with
to
be
and
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is
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syntrophic
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&
A
the
to
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could
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the
substrate.
Ready
resided
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N
for
of
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handbook
for
some
for missing
degradation
may
ethanol
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(1992)
true the
as
such
which
edition.
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the
the
be
the
exploring
opportunities
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yet
of
before
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association
degradation
in
extent
expected
identity
not
same
metabolic
organic
relationships
in
organisms
contrast,
The
unclear.
the
methanogens. The
information
factor
Arch.
&
enrichment
on
(1979)
Vol
association
speaks
process
M.
resemble
potential
time,
anaerobic
assigning
the
of
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metabolic
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15
active
R.
matter conditions
Microiol.
of
is
to
Anaerobic
lactate
The
with
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biology
enrichments
Springer-Verlag.
activities.
the
to
from
for
the
of
tested,
be
ethanol
independent
with
about
in
syntrophic
growth
for
difference
the
organic for
rare.
observed,
M
the
syntrophy
way
organisms
Despite
Choline
the
45:
them
122:
(1977)
or
relationships
or
local
H2-utilizing
of
maripaludis
(salt SRBs
and
high
bacterium
sea
and
circumstances
Yet,
161- substrate
ethanol
of
of
129-1
bacteria:
of
to
bedthe
matter
the
life.
SRBs from
Growth
water a
using
by
lactate
SRB2
169.
we
in
metabolic
of
interactions
using
two
can
secondary
the
35.
involved
results
a
salinity
pp
in
hydrogen
observed
that Coculture
Ceder
disappearance
methanogenic
to
role
to
enrichments
be
under
ecophisiology,
sulfate
between
of
under and
277-299.
marine
aspects
may
interact
samples
degrades
accommodate
Desulfovibrio
of
expected
observed
SRB3
prevailing
between
Swamp
sulphate
versatility
in
not
product
anaerobic,
our
in
the
of
reducer
removal.
settings,
the
syntrophically
In:
are
SRB2
environments
be
DesulJbvibrio
fatty
experimental
from
on
potential
bacteria.
for
to
syntrophic
dynamics,
Balows
carmot
present
reduction.
(SRB3),
the
glutamate
isolation, worth
from
acids
in
SRB abound,
in
and
for
group.
marine
of
native
fresh
where
When
the
the
the
in
be
for
et
the
M
as
in
of a
methanogenesis
Winfrey
J.
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And
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50
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of
589-
cooperation
of
vulgaris
sulfate
16
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on
during
and
Microbiol.
carbon
John
in
sulfur-reducing
methanogenic
Wiley
coculture
and
interspecies
33:
electron
&
275-28
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metabolism
bacteria.
degradation.
flow
Inc.,
1.
H2
New
during
transfer
pp
of
469-5
York.
Microbioly
acetate
microbial
between
85.
In: or