Alternative Pathways for Hydrogen Disposal During Fermentation in the Human Colon

Abstract

Hydrogen gas, which is produced during fermentation in the human colon, is either excreted in breath or metabolised by gut bacteria through a variety of pathways. These may include methanogenesis, dissimilatory sulphate reduction, and acetogenesis. To determine which of these routes predominates in the large intestine, stools were taken from 30 healthy subjects and incubated as 5% (w/v) slurries with Lintner's starch. In 23 of 30 subjects, methane production was the main method of hydrogen disposal. In the remaining seven, high rates of sulphate reduction were recorded together with raised production of H2S. All samples showed relatively low rates of hydrogen evolution and of acetate formation from CO2 and H2. Sulphate reduction and methanogenesis seem to be mutually exclusive in the colon and this is probably linked to sulphate availability. Sulphate reduction, methanogenesis, and acetogenesis were strongly influenced by pH. Sulphate reduction was optimal at alkaline pH values whereas methane production was maximal at a neutral pH and acetogenesis favoured acidic conditions. Faecal H2S values were related to carriage of sulphate reducing bacteria. These data show that a number of competing pathways for hydrogen disposal are possible in the large gut and that a variety of factors such as colonic pH and sulphate availability can determine which of these mechanisms predominates.

Full text

Gut,
1990,31,679-683
Alternative
pathways
for
hydrogen
disposal
during
fermentation
in
the
human
colon
G
R
Gibson,
J
H
Cummings,
G
T
Macfarlane,
C
Allison,
I
Segal,
H H
Vorster,
A
R
P
Walker
Abstract
Hydrogen
gas,
which
is
produced
during
fermentation
in
the
human
colon,
is
either
excreted
in
breath
or
metabolised
by
gut
bacteria
through
a
variety
of
pathways.
These
may
include
methanogenesis,
dissimilatory
sulphate
reduction,
and
acetogenesis.
To
determine
which
of
these
routes
predominates
in
the
large
intestine,
stools
were
taken
from
30
healthy
subjects
and
incubated
as
5%
(w/v)
slurries
with
Lintner's
starch.
In
23
of
30
subjects,
methane
production
was
the
main
method
of
hydrogen
disposal.
In
the
remaining
seven,
high
rates
of
sulphate
reduction
were
recorded
together
with
raised
production
of
H2S.
All
samples
showed
relatively
low
rates
of
hydrogen
evolution
and
of
acetate
formation
from
CO2
and
H2.
Sulphate
reduction
and
methanogenesis
seem
to
be
mutualiy
exclusive
in
the
colon
and
this
is
probably
linked
to
sulphate
availability.
Sulphate
reduction,
methanogenesis,
and
acetogenesis
were
strongly
influenced
by
pH.
Sulphate
reduction
was
optimal
at
alkaline
pH
values
whereas
methane
production
was
maximal
at
a
neutral
pH
and
acetogenesis
favoured
acidic
condi-
tions.
Faecal
H2S
values
were
related
to
carriage
of
sulphate
reducing
bacteria.
These
data
show
that
a
number
of
competing
path-
ways
for
hydrogen
disposal
are
possible
in
the
large
gut
and
that
a
variety
of
factors
such
as
colonic
pH
and
sulphate
availability
can
determine
which
of
these
mechanisms
predominates.
The
aerobic
metabolism
of
carbohydrate
in
mammalian
cells
requires
oxygen
as
the
terminal
electron
acceptor
and
produces
carbon
dioxide,
water,
and
energy
as
the
principal
end
products.
In
anaerobic
systems
such
as
the
lumen
of
the
human
colon,
however,
starch,
non-starch
poly-
saccharides
(dietary
fibre),
and
other
substrates
are
fermented
by
the
resident
microflora
to
yield
short
chain
fatty
acids,
carbon
dioxide,
hydrogen,
and
energy.
'
Molecular
hydrogen
is
used
by
intestinal
methanogenic
bacteria
in
many
animal
species
to
reduce
carbon
dioxide
to
methane.
Methano-
TABLE
I
Effect
of
different
storage
treatments
upon
hydrogen
production
and
sulphate
reducing
activity
in
faeces.
Values
are
mean
(SEM)
Rate
of
hydrogen
Rate
of
sulphate
accumulation*
reductiont
Viable
count
SRB
Treatment
(nmollg
dry
wt/d)
(nmol/g
dry
wt/d)
(loglo/gdrywtfaeces)
Fresh
faeces
72-5
(13-2)
321-8
(36
4)
9
0
(1-6)
Ambient
temperature
for
24
h
54
6
(11-6)
281-2
(29
4)
9
0
(1-2)
Freezing
in
10%
w/v
glycerol
3
5
(0-9)
146-7
(28
4)
6-0
(1-7)
*9
hour
incubation;
t48
hour
incubation.
SRB
=sulphate
reducing
bacteria.
genic
bacteria
have
been
identified
in
man2
3
but
population
studies
have
shown
that
as
few
as
21%
(Indian
adults)
or
as
many
as
95%
(rural
black
African
teenagers)
excrete
methane
in
breath.4
Similarly,
there
is
great
variation
in
the
produc-
tion
of
hydrogen
when
subjects
are
challenged
with
standard
doses
of
fermentable
carbohydrate
such
as
lactulose.5
Sulphate
reducing
bacteria
are
a
group
of
obligately
anaerobic
bacteria
which
utilise
sulphate
as
an
oxidising
agent
for
the
dissimila-
tion
of
organic
matter
-
that
is,
replacing
oxygen
in
conventional
aerobic
respiration.
Molecular
hydrogen
can
also
act
as
an
electron
donor
for
dissimilatory
sulphate
reduction.6
The
major
end
product
of
this
process
is
sulphide,
which
is
rapidly
hydrolysed
to
H2S.
In
vitro
studies
have
shown
that
in
the
large
gut,
sulphate
reducing
bacteria
can
outcompete
methanogenic
bacteria
for
limited
amounts
of
hydrogen
if
sufficient
sulphate
is
available.7
A
third
role
whereby
molecular
hydrogen
can
be
metabolised
in
the
colon
is
by
the
reduction
of
carbon
dioxide
to
acetate,
or
homoacetogenesis.5
Thus,
a
number
of
pathways
exist
for
the
disposal
of
reducing
equivalents
formed
during
colonic
fermentation.
Competition
for
hydrogen
among
methanogenic
bacteria,
sulphate
reducing
bacteria,
acetogenic
bacteria,
and
other
species
probably
occurs.
This
is
important
to
man
since
removal
of
hydrogen
by
the
large
intestinal
microflora
shifts
fermentation
to
more
oxidized
end
products
so
increasing
energy
yield.9
Little
is
known
about
these
processes
in
the
colon.
We
therefore
measured
rates
of
hydrogen
accumula-
tion,
sulphate
reduction,
methanogenesis,
and
acetogenesis
in
faecal
samples
from
30
subjects
to
determine
whether
a
competitive
relation
exists
between
these
processes.
Methods
SUBJECTS
Fresh
faecal
samples
were
collected
from
30
healthy
volunteers
(15
men
and
15
women)
aged
28-60
years.
Twenty
samples
were
taken
from
African
subjects
in
the
village
of
Hekpoort,
Western
Transvaal
and
10
samples
were
from
staff
of
the
South
African
Institute
of
Medical
Research
in
Johannesburg.
Faeces
were
col-
lected
in
the
morning
before
being
flown
over-
night
to
London
for
processing
in
Cambridge
the
following
afternoon.
No
subject
had
taken
anti-
biotics
for
at
least
eight
weeks
before
giving
a
specimen.
Samples
were
transported
at
ambient
temperature
as
initial
studies
showed
that
freez-
ing
the
faeces
in
10%
(w/v)
glycerol
significantly
decreased
hydrogen
production
and
sulphate
reducing
activity
(Table
I).
MRC
Dunn
Clinical
Nutrition
Centre,
Cambridge
G
R
Gibson
J
H
Cummings
G
T
Macfarlane
C
Allison
Baragwanath
Hospital,
University
of
the
Witwatersrand,
Johannesburg,
South
Africa
I
Segal
Department
of
Physiology,
Potchefstroom
University,
Potchefstroom,
South
Africa
H H
Vorster
South
African
Institute
for
Medical
Research,
Johannesburg,
South
Africa
A
R
P
Walker
Correspondence
to:
Dr
G
R
Gibson,
MRC
Dunn
Clinical
Nutrition
Centre,
100
Tennis
Court
Road,
Cambridge
CB2
1QL.
Accepted
for
publication
4
September
1989
679

Gibson,
Cummings,
Macfarlane,
Allison,
Segal,
Vorster,
Walker
BREATH
METHANE
Duplicate
end-expiratory
breath
samples
were
collected
into
20
ml
plastic
syringes
and
methane
was
determined
by
gas
chromatography
using
a
Pye
Unicam
PU-4500
with
flame
ionisation
detector.
Samples
of
room
air
were
also
taken
and
the
value
subtracted
from
the
breath
sample.
A
methane
producer
was
defined
as
a
subject
with
more
than
1
ppm
methane
in
breath
above
values
in
ambient
air.
The
study
was
approved
by
the
Ethical
Com-
mittee
of
the
Medical
School
of
the
University
of
the
Witwatersrand,
Johannesburg.
FAECAL
SLURRIES
Faecal
slurries
(5%
w/v)
were
prepared
by
homo-
genising
samples
in
anaerobic
sodium
phosphate
buffer
(0
1
mol/l,
pH
7
0).
For
measurements
of
hydrogen
production
and
methanogenic,
sul-
phate
reduction,
and
acetogenic
rates,
Lintner's
starch
(0-2%
w/v)
was
incorporated
into
the
slurries
as
the
fermentable
substrate.
METHANE
FORMATION
RATES
Methane
production
from
faecal
slurries
was
measured
as
described
by
Allison
and
Macfarlane.'0
The
linear
part
of
the
methane
production
plot
during
a
48
hour
incubation
period
was
used
to
calculate
the
rate
of
methano-
genesis.
SULPHATE
REDUCTION
RATES
Triplicate
sub-samples
(5
ml)
were
removed
from
each
faecal
slurry
and
5
,ul
volumes
of
carrier
free
sodium
(35S)
sulphate
added.
Samples
were
then
incubated
anaerobically
for
18
hours
at
37°C
before
freezing
and
subsequent
distillation
using
the
method
of
J0rgensen."
Rates
of
sulphate
reduction
were
calculated
from
the
amount
of
acid
volatile
('5S)
sulphide
formed."2
ACETATE
FORMATION
RATES
Rates
of
acetogenesis
in
5
ml
sub-samples
from
faecal
slurries
were
determined
in
triplicate
using
the
method
of
Jones
and
Simon.'3
ENUMERATION
OF
SULPHATE
REDUCING
BACTERIA
Viable
populations
of
sulphate
reducing
bacteria
were
counted
using
the
agar
shake
dilution
method
with
acetate,
lactate,
propionate,
butyrate,
and
H2/CO2
as
electron
donors6
since
various
studies6
14
15
have
shown
that
these
are
the
major
substrates
that
support
the
growth
of
sulphate
reducing
bacteria.
Dilution
tubes
were
incubated
anaerobically
at
37°C
for
14
days.
After
this
time,
growth
of
sulphate
reducing
bacteria
was
indicated
by
a
precipitation
of
ferrous
sulphide
and
the
number
of
black
colonies
formed
was
counted.
ISOLATION
AND
CHARACTERISATION
OF
SULPHATE
REDUCING
BACTERIA
Single
colonies
were
removed
from
the
highest
agar
shake
dilution
tubes
and
subcultured
into
liquid
media.
Pure
cultures
were
obtained
by
successive
passage
through
agar
shakes
and
sulphate
reducing
bacteria
were
characterised
using
the
criteria
of
Keith
et
al.16
HYDROGEN
SULPHIDE
CONCENTRATIONS
Values
of
H2S
in
faeces
were
measured
(after
precipitation
of
sulphides
in
10%
w/v
zinc
acetate)
using
the
spectrophotometric
method
of
Cline.
7
EFFECT
OF
pH
UPON
METHANOGENESIS,
DISSIMILATORY
SULPHATE
REDUCTION,
AND
ACETOGENESIS
IN
FAECAL
SLURRIES
To
test
whether
colonic
pH
could
significantly
influence
rates
of
methanogenesis,
sulphate
reduction,
and
acetogenesis,
faecal
slurries
were
prepared
as
described
and
adjusted
to
a
range
of
pH
values
(5
5-8
5
in
0
5
increments).
Methano-
genic
and
acetogenic
rates
were
calculated
as
before
and
sulphate
reducing
activity
was
deter-
mined
by
production
of
H25.
Results
TRANSPORT
OF
FAECAL
SAMPLES
Two
approaches
were
tested
to
assess
the
most
favourable
method
of
transporting
faecal
samples
to
the
UK
for
processing.
Faeces
incubated
at
ambient
temperature
for
24
hours
under
an
atmosphere
of
oxygen-free
nitrogen
as
well
as
samples
frozen
in
a
slurry
(5%
w/v)
containing
10%
w/v
glycerol
were
prepared.
Rates
of
hydrogen
production
and
sulphate
reducing
activity
were
subsequently
determined
and
compared
with
those
found
in
fresh
faeces.
Data
presented
in
Table
I
show
that
activities
were
always
reduced
in
the
incubated
and
frozen
samples.
The
percentage
inhibition
of
hydrogen
release,
sulphate
reduction,
and
numbers
of
viable
sulphate
reducing
bacteria,
however,
were
appreciably
greater
in
the
frozen
samples.
Faeces
were
therefore
transported
from
South
Africa
at
ambient
temperature
in
sealed
plastic
bags.
TABLE
II
Hydrogen
metabolism
infaecal
samples
from
30
healthy
subjects.
Results
are
mean
(SEM)
Breath
Rate
of
hydrogen
Rate
of
methane
Rate
of
sulphate
Rate
of
Amount
of
methane
accumulation*
productiont
reductiont
acetogenesist
H2S
in
faeces
No
(ppm)
(nmollg
dry
wtld)
(nmol/g
dy
wtld)
(nmol/g
dry wtld)
(nmol/g
dry
wt/d)
(mM)
GroupA
23
20-1(3-7)
162(25-1)
7902(1061)
92(43)
590(84)
0-05(0-01)
Group
B
7
0
154(32-1)
0
1478
(661)
108
(14-3)
0-21(0-03)
Group
A=number
of
sulphate
reducing
bacteria
<
10'/g
dry
wt
faeces.
Group
B=number
of
sulphate
reducing
bacteria
>
107/g
dry
wt
faeces.
*9
hour
incubation;
t48
hour
incubation.
680

Alternative
pathwaysfor
hydrogen
disposal
duringfermentation
in
the
human
colon
*
10-78
TABLE
III
Percentage
distribution
of
sulphate
reducing
bacteria
(SRB)
in
human
faecal
samples.
Results
are
mean
(SEM)
Percentage
SRB
utilising
Carbon
source
each
substrate
Acetate
14-1
(7-0)
Lactate
63-1
(24-1)
Propionate
9-4
(2
3)
Butyrate
9-2
(2-8)
H2/CO2
4-3
(1-6)
0
998
*
881
9.59
*
98
0102
*
9-78
*
578
0-10
*0
-
*
6.04
*0
*
5-25
bO..
*
6-79
SRB
SRB
+ve
-ve
Figure
1:
Viable
counts
of
sulphate
reducing
bacteria
(SRB)
in
relation
to
hydrogen
sulphide
values
in
human
faeces.
Numbers
indicate
logl0
SRB/g
dry
weight
faeces.
HYDROGEN
ACCUMULATION,
METHANOGENESIS,
SULPHATE
REDUCTION/AND
ACETOGENESIS
IN
FAECAL
SLURRIES
When
faecal
slurries
were
incubated
with
Lintner's
starch
a
small
amount
of
hydrogen
accumulated
during
the
first
nine
hours
of
incubation.
The
rate
of
hydrogen
release
during
this
time
did
not
differ
greatly
in
any
of
the
samples
tested
(Table
II).
After
nine
hours,
hydrogen
concentrations
gradually
declined.
To
determine
the
route
of
hydrogen
uptake
during
this
period,
rates
of
methanogenesis,
sulphate
reduction,
and
acetogenesis
were
measured
and
compared
in
each
of
the
faecal
slurries.
On
the
basis
of
methanogenesis
rates
and
numbers
of
sulphate
reducing
bacteria
(SRB)
in
faeces,
the
subjects
divided
readily
into
two
groups
(Table
II).
Most
subjects
(group
A;
n=
23)
shared
high
rates
of
faecal
methanogenesis
and
had
less
than
107
SRB/g
dry
weight
faeces.
In
group
A,
21
of
the
23
subjects
had
methane
in
the
breath.
None
of
the
subjects
in
group
B
(n=7)
had
methane
in
the
breath,
produced
methane
in
vitro,
or
had
more
than
107
SRB/g
of
faeces.
Group
B
subjects
had
high
rates
of
sulphate
reduction
in
faeces
and
higher
concentrations
of
sulphide.
Low
rates
of
sulphate
reduction
and
H2S
formation
were
detected
in
some
samples
from
group
A
but
these
were
much
less
than
those
measured
in
the
group
B
subjects
(Table
II).
Viable
populations
of
sulphate
reducing
bacteria
were
enumerated
with
acetate,
lactate,
propionate,
butyrate,
and
H2/CO2
as
electron
donors
to
give
total
counts
of
faecal
sulphate
reducing
bacteria.
Sulphate
reducer
counts
showed
a
strongly
positive
association
with
H2S
concentrations
in
faeces
(Fig
1).
Hydrogen
sulphide
values
in
the
four
group
A
faecal
samples
that
contained
less
than
107
SRB/g
were
similar
to
those
in
which
sulphate
reducing
bacteria
were
completely
absent.
Rates
of
acetogenesis
were
relatively
low
in
all
samples
tested
(Table
II).
Those
in
group
B,
however,
were
approximately
double
those
found
in
group
A
suggesting
that
acetate
produc-
TABLE
IV
Effect
of
pH
upon
methanogenesis,
sulphate
reduction,
and
acetogenesis
in
faecal
slurries.
Results
are
mean
(SEM)
of
triplicate
determinations
Rate
of
Rate
of
H2S
Rate
of
methanogenesis
production
acetogenesis
pH
(nmollg
dry
wt/h)
(nmol/g
dry
wt/h)
(nmol/g
dry
wtlh)
5-5
10-2
(3
2)
3-8
(0-7)
1-84
(0-04)
6-0
31*2(8-1)
11
9(62)
1-93(006)
6
5
43-6
(7
1)
13-4
(5
3)
4-65
(0-8)
7-0
57-2(8-9)
25
7(5
3)
0-83(0-1)
7.5
54
2
(9
4)
28-9
(4
1)
0-69
(0-1)
8-0
44-3
(5-2)
27-4
(4 9)
0-28
(0-09)
8
5
21-4(3-1)
10-4(2-7)
0
tion
from
H2/CO2
may
be
more
important
in
non-methanogenic
subjects.
ISOLATION
AND
CHARACTERISATION
OF
SULPHATE
REDUCING
BACTERIA
The
highest
numbers
of
sulphate
reducing
bacteria
were
found
using
lactate
as
a
source
of
carbon
and
energy
(Table
III).
The
dominant
bacteria
consisted
of
curved
rods
of
various
sizes,
identified
as
belonging
to
the
genus
Desulfovibrio.
18
Desulfovibrio
species
do
not
have
a
complete
TCA
cycle
and
acetate
is
there-
fore
a
normal
end
product
of
their
metabolism.
As
a
consequence,
coccobacillary
rods,
identified
as
the
acetate
oxidizing
Desulfobacter
species'920
were
occasionally
isolated
from
lactate-
containing
tubes.
In
dilution
tubes
containing
acetate
as
the
sole
carbon
source,
Desulfobacter
species
were
always
numerically
predominant.
When
propionate
was
the
electron
donor,
the
major
bacteria
isolated
belonged
to
the
genus
Desulfobulbus2'
which
incompletely
oxidizes
propionate
to
acetate
and
carbon
dioxide.
Large
non-spore
forming
rod
shaped
bacteria
identified
as
Desulfotomaculum
acetoxidans22
23
were
isolated
from
butyrate
enrichments
with
Desulfovibrio
species
and
Desulfobulbus
species
generally
present
with
H2/CO2
as
a
substrate.
INFLUENCE
OF
pH
UPON
METHANOGENIC,
SULPHATE
REDUCING,
AND
ACETOGENIC
ACTIVITY
Data
presented
in
Table
IV
show
that
the
optimal
pH
values
for
methanogenesis
and
dis-
similatory
sulphate
reduction
were
7-0
and
7-5
respectively.
At
acidic
pH
values
both
processes
were
substantially
inhibited
whereas
homoaceto-
genesis
was
highest
at
relatively
low
pH
values.
Discussion
Dietary
carbohydrate
that
escapes
digestion
and
absorption
in
the
small
bowel
passes
into
the
caecum
where
fermentation
by
anaerobic
bacteria
occurs
producing
hydrogen.24
A
number
of
fates
for
this
hydrogen
then
exist.
A
small
proportion
may
pass
through
the
gut
wall
into
the
blood
and
be
transported
to
the
lungs
where
it
is
then
excreted
in
breath.2526
Alternatively,
hydrogen
can
be
metabolised
by
the
large
intestinal
microflora.
The
removal
of
hydrogen
allows
a
depletion
of
electron
sink
products
such
as
lactate,
succinate,
and
ethanol
resulting
in
a
higher
energy
yield
from
fermentation.9
Thus
adequate
removal
of
H2
allows
a
more
complete
recovery
of
energy
by
bacteria
from
the
degrada-
tion
of
organic
substrates.
An
appreciable
pro-
portion
of
hydrogen
may
be
removed
by
the
action
of
methanogenic,27
sulphate
reducing,28
and
homoacetogenic
bacteria.2930
All
three
pro-
cesses
remove
four
moles
of
hydrogen
per
mole
CO2
or
SO2
reduced.
Hydrogen
is
essential
for
the
growth
of
colonic
methanogens2
3I31
and
if
this
substrate
is
removed
methanogenesis
cannot
occur.
In
the
present
study,
considerable
methane
production
occur-
red
only
when
sulphate
reducing
bacteria
were
not
active
-
that
is
group
A
subjects
(Table
II).
E
E
r-
<n
03"
CN
4--
I
0
c
02
o2
cJ
C
C
0
0
681

Gibson,
Cummings,
Macfarlane,
Allison,
Segal,
Vorster,
Walker
The
metabolic
end
product
of
dissimilatory
sulphate
reduction
(H2S)
is
thought
to
be
toxic
to
methanogenic
bacteria,32
34but
at
the
low
concen-
trations
measured
in
faeces
(Fig
1),
it
will
not
exert
any
direct
inhibitory
effect.
When
sulphate
is
available,
sulphate
reducing
bacteria
are
known
to
have
a
higher
substrate
affinity
for
hydrogen
than
methanogenic
bacteria,35-
and
this
is
a
more
likely
explanation
for
the
apparent
mutual
exclusion
of
methanogenesis
and
sul-
phate
reduction
in
the
large
gut.
We
have
shown
that
sulphate
reducing
bacteria
outcompete
methanogenic
bacteria
for
hydrogen
when
faecal
slurries
from
methane
and
non-methane
produc-
ing
subjects
are
mixed
together.7
In
the
group
A
samples,
low
rates
of
sulphate
reduction
and
H2S
production
occurred
even
in
the
presence
of
active
methanogenesis.
It
is
likely
that
the
small
amount
of
sulphate
reduced
is
by
assimilation
into
sulphur-containing
amino
acids
and
subsequently
proteins.
The
H2S
produced
in
faeces
from
group
A
subjects
was
probably
released
from
these
amino
acids
during
protein
fermentation.
Four
of
the
group
A
subjects
had
low
numbers
of
sulphate
reducing
bacteria
in
faeces
but
H2S
values
were
similar
to
those
found
in
group
B
samples,
so
these
sulphate
reducers
were
active
at
values
that
did
not
affect
hydrogen
uptake
by
methanogenic
bacteria.
Some
sulphate
reducing
bacteria
can
grow
fermentatively
in
the
absence
of
sulphate28
and
in
this
case
methano-
genic
bacteria
may
act
as
important
hydrogen
scavengers
to
keep
concentrations
below
thermo-
dynamically
unfavourable
values.3839
Sulphate
reducing
bacteria
would
then
function
as
hydro-
gen
producing
acetogens.29
An
alternative
route
for
hydrogen
disposal
is
by
reduction
of
CO2
to acetate.
A
recent
study
has
indicated
that
this
may
occur
in
man.8
Homoacetogenesis
involves
the
utilisation
of
hydrogen
and
carbon
dioxide
to
form
acetate
via
acetyl
CoA.40
Homoacetogenic
bacteria
are,
how-
ever,
competitively
displaced
by
methanogenic
bacteria
for
available
hydrogen
in
other
anaerobic
ecosystems.4142
Thus,
these
bacteria
will
only
become
active
when
there
is
little
hydrogen
uptake
by
sulphate
reducing
or
methanogenic
bacteria,
explaining
the
low
rates
of
acetogenesis
recorded
in
this
study.
The
fact
that
rates
were
generally
higher
in
the
non-methanogenic
(group
B)
slurries
may
be
linked
to
the
concentra-
tion
of
available
sulphate.
If
sulphate
is
limited
and
hydrogen
is
in
relative
excess,
some
of
the
hydrogen
remaining
after
sulphate
reduction
could
then
be
available
for
metabolism
by
homo-
acetogenesis
(some
other
unknown
factor
would
have
to
limit
methanogenesis,
however).
The
concentration
of
sulphate
present
in
the
large
gut
is
therefore
critical
for
determining
which
of
these
processes
occurs.
If
sufficient
sulphate
exists,
sulphate
reducing
bacteria
will
predominate.
If
the
colonic
sulphate
pool
is
low,
however,
these
bacteria
will
not
utilise
appreci-
able
amounts
of
hydrogen.
During
these
condi-
tions,
methanogenic
bacteria
or
perhaps
aceto-
genic
bacteria
will
become
important.
Recent
studies
have
shown
that
a
large
variability
in
the
amount
of
sulphate
reaching
the
colon
exists.43
The
contribution
of
endogenous
sulphate
is
approximately
1
mmol/d
whereas
dietary
levels
Figure
2:
Postulated
mechanisms
for
hydrogen
disposal
in
the
human
colon.
SRB=sulphate
reducing
bacteria;
MB=methanogenic
bacteria;
AB=acetogenic
bacteria;
VFA=volatile
fatty
acids.
Carbohydrate
which
reaches
the
colon
is
fermented
by
populations
of
anaerobic
bacteria
producing
VFA
and
the
gases
H2
and
CO2.
A
small
proportion
of
the
H2
is
excreted.
The
remainder
can
then
undergo
further
metabolism.
If
sufficient
sulphate
exists,
SRB
are
primarily
responsible
and
H2S
is
produced.
During
conditions
of
low
sulphate
availability,
however,
MB
and
AB
are
able
to
combine
H2
with
C02
to
form
methane
and
acetate
respectively.
can
range
from
2-16
mmol/d,
with
maximal
absorption
occurring
below
7
mmol/d.
Colonic
pH
may
also
be
an
important
factor
controlling
the
rate
of
hydrogen
uptake
in
the
large
intestine.
The
right
colon,
where
most
carbohydrate
fermentation
occurs,
is
a
region
of
low
pH
whereas
conditions
in
the
left
colon
and
sigmoid
rectum
areas
frequently
approach
neutrality.'
Homoacetogenesis
may
become
important
at
low
pH
in
the
colon,
because
in
vitro
studies
showed
that
faecal
sulphate
reduc-
ing
and
methanogenic
bacteria
were
relatively
pH-sensitive,
preferring
an
environment
that
is
neutral
or
slightly
alkaline,
whereas
highest
rates
of
acetogenesis
occurred
at
acidic
pH
values
(Table
IV).
Furthermore,
we
have
shown
previously
using
a
three
chambered
fermenta-
tion
system
that
at
a
pH
of
6X0,
hydrogen
uptake
can
occur
without
any
appreciable
contribution
from
sulphate
reducing
or
methanogenic
bacteria.45
A
number
of
possible
pathways
for
disposal
of
H2
exist
therefore
in
man
and
are
summarised
in
Figure
2.
What
are
the
clinical
consequences
of
this?
Firstly,
it
makes
it
highly
unlikely
that
simple
relations
can
be
drawn
between
fermenta-
tion
of
specified
substrates,
such
as
lactulose,
and
H2
evolution
in
breath.
In
practice,
widely
differing
responses
to
standard
oral
doses
of
fermentable
carbohydrate
are
seen
among
sub-
jects.5
Bjorneklett
and
Jenssen46
have
shown
that
subjects
who
produce
methane
during
fermenta-
tion
produce
appreciably
less
H2
in
breath
in
response
to
a
standard
dose
of
lactulose.
Secondly,
if
H2
is
not
further
metabolised,
fermentation
may
be
incomplete
and
intermedi-
ates
such
as
lactate,
succinate,
and
ethanol
are
likely
to
accumulate.
D-lactate,
produced
by
colonic
bacteria,
is
only
partly
metabolised
in
man
and
can
cause
severe
metabolic
disturbance
on
occasions.47
Thirdly,
if
H2
gas
is
not
metabo-
lised
the
volume
of
gas
accumulating
in
the
gut
will
be
substantially
greater
than
if
CH4
is
produced
because
the
reaction:
C02+4H2-)CH4+2H20
Carbohydrate
Excretion
(breath)
SB
H25
Fermentative
HsCHCOO
bacteria
H2
AB
H3CH
C02
MB
CH4
VFA
682

Alternative
pathwaysfor
hydrogen
disposal
duringfermentation
in
the
human
colon
683
reduces
five
volumes
of
gas
to
one.
Fourthly,
the
end
products
of
these
various
terminal
oxidative
reactions
differ
in
their
toxicity.
Methane
is
a
harmless
gas,
readily
expelled.
Acetate
is
absorbed
and
metabolised
by
peripheral
tissue
such
as
muscle
but
H2S
is
highly
toxic
and
may
poison
colonic
epithelial
cells
if
not
oxidised
rapidly
after
absorption.
The
capacity
for
high
rates
of
H2S
production
exists
in
some
people
(Table
II)
and
it
may
be
that
sulphate
reducing
bacteria
play
a
part
in
the
aetiology
of
some
large
gut
disorders.
We
have
previously
shown,7
using
in
vitro
faecal
slurries,
that
up
to
3
mM
H2S
may
be
produced
during
a
48
hour
incubation.
In
this
study
faecal
H2S
did
not
rise
above
a
concentra-
tion
of
04
mM
(Fig
1).
This
suggests
that
some
detoxification
mechanism
for
H2S
is
operative
in
the
large
gut.
Such
a
mechanism
may
include
incorporation
into
sulphide
containing
amino
acids
or
the
production
of
mercaptans
-
for
example,
mercaptoacetate
or
mercaptobutyrate.
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Cummings
JH.
Fermentation
in
the
human
large
intestine:
evidence
and
implications
for
health.
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1985;
i:
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TL,
Wolin
MJ,
Macario
EC
de,
Macario
AJL.
Isolation
of
Methanobrevibacter
smithii
from
human
feces.
Appl
Env
Microbiol
1982;
43:
227-32.
3
Miller
TL,
Wolin
MJ.
Stability
of
Methanobrevibacter
smithii
populations
in
the
microbial
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excreted
from
the
human
large
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Env
Microbiol
1983;
45:
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I,
Walker
ARP,
Lord
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1988;
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1988;
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GR,
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GT,
Cummings
JH.
Occurrence
of
sulphate-reducing
bacteria
in
human
faeces
and
the
relation-
ship
of
dissimilatory
sulphate
reduction
to
methanogenesis
in
the
large
gut.
]
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1988;
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JH,
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bacteria
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methano-
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1988;
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MJ.
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C)
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MJ.
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methane-
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N,
eds.
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methane
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slurries
of
human
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Gen
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1988;
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the
quantification
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20
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sulfate-reducing
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D.
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N.
Studies
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dissimilatory
sulfate-
reducing
bacteria
that
decompose
fatty
acids.
Incomplete
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propionate
by
Desulfobulbus
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sp.
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1982;
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22
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F,
Pfennig
N.
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sporing
acetate-
oxidizing
sulfate-reducing
bacterium,
Desulfotomaculum
acetoxidans
(emend).
Arch
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1977;
112:
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23
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F,
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N.
Sporulation
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1981;
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Calloway
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1966;
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