Vol. 79, No. 1, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
METHANE PRODUCTION BY CELL-FREE
PARTICULATE
FRACTION OF RUMEN BACTERIA
F.D. Sauer, R.S. Bush, S. Mahadevan and J.D. Erfle Animal Research Institute, Agriculture Canada, Ottawa, Ontario, Canada, KlA OC4 .
Received
September
21,197i'
retained methanogenic activity when stored at -60" SUMMARY: Rumen bacteria under H2. This activity, which resides in Methanobacterium ruminantium and Methanobacterium mobilis, is not lost when the cells are broken, as has been suggested. Unlike in Methanosarcina barkerii and Methanobacterium M.o.H., in rumen bacteria methanogenic enzymes are not soluble but readily precipitated at 15,000 g. Methane was synthesized from tetrahydrofolate derivatives but at slower rates than from C02. From the data, it was not possible to determine if methyland methylene tetrahydrofolate were oxidized to CO2 prior to reduction to CH4. In room light, CH3-Bl2 was reduced to CH4 non-enzymatically in the presence of protein. When the reactions were carried out in the dark, very little CH4 was formed from CH3-Bl2 by rumen bacterial enzymes. The cell-free particulate fraction did not require added ATP for methanogenesis but showed an absolute requirement for H2. INTRODUCTION: hydrogen
Most methane
and carbon
Methanosarcina (2).
are
oscillation at 30,000
can utilize
out with
prepared
and French
pressure
cell
ruminantium
(approx. (5,
and stored
fraction
after
production
for
the pathway
M.o.H.
and fi.
oscillation
(3)
disruption
(4)
(1).
with
In addition,
growth
and methane
of methane
formation Cell-free
barkerii.
or by combined
followed
sonic
by centrifugation
25 to 60 min.
production
report
with
requirements
and carbon
and acetate
Methanobacterium by sonic
flora
In this
dealing
nutritional
energy
methanol
prepared
Methanobacterium
methane
both
generally
g for
the rumen
have simple
supplying
Much of the work
has been carried extracts
dioxide
barkerii
production
bacteria
4 x lo8 6).
complete
of methane
cells
We have
as described
we show that
and Methanobacterium per ml)
observed
previously
methanogenic
cell
by the membrane
and together that
(7),
account
total
methane.
in the particulate
of intermediates
of rumen bacterial
for
in
pellets,
synthesize
is entirely
The role fraction
are present
rumen bacterial actively
activity
disruption.
mobilis
in the cells
was
examined.
Copyright All rights
0 1977
by Academic Press, Inc. in any form reserved.
of reproduction
124 ISSN
0006-291
X
Vol. 79, No. 1, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MATERIALS AND METHODS: Preparation of cell-free extract. Rumen bacterial pellets were prepared as described (7) but stored at -60" in clarified rumen fluid under HT. To thawed 10 ml pellets (ZOO to 250 mg protein) was added with continuous H; gassing: FAD (1 mM); CoA, (0.1 mM); pyruvate, (5 mM); and pyruvate lyase isolated from Clostridium pasteurianum (8) (' 2 mg). These additions kept the bacterial cell fractions fully reduced through subsequent steps provided that extreme care was used to exclude 02 and maintain a constant N2 or H2 atmosphere. Cells were disrupted by 3 min sonication in a Bronson sonifier cell disruptor (90 to 100 watts) in cycles of 15 s on and 45 s off. The partially disrupted cell suspension was then put through an R-l Ribi cell fractionator at 25,000 p.s.i. This was followed by an additional 3 min sonication as before. The cell extract was then centrifuged 30 min at 2380 g (unless specified otherwise) under H2 in tightly stoppered tubes. A one ml aliquot of this cell extract was centrifuged at 100,000 g. The resulting pellet was fixed in glutaraldehydejosmium tetraoxide and stained with uranyl acetate/lead citrate for examination in a Phillips 300 electron microscope. No intact bacterial cells were found. Incubation and assay procedure. Cell fractions (0.5 ml) were incubated at 38' in 0.1 M potassium phosphate buffer (pH 7.0) containing 1 mM Na2S.9H20 (final vol. 1.0 ml) in 5 ml Erlenmeyer flasks sealed with serum bottle rubber stoppers. Additions were made as specified in legends to Figures and Tables. Before addition of enzyme, each flask was evacuated and flushed with Hz for three 1 min periods. Substrate and enzyme were added by microsyringe to Hz filled flasks before incubation. Gas samples were withdrawn at timed intervals with a gas syringe (Precision Sampling Corp., Baton Rouge, La.) and assayed on a gas partitioner (Fisher Model 1200) coupled to a recording integrator (Infotronics CRS 1lOA). The effluent gas, after partitioning, was passed through copper tubing into a combustion furnace and the;t4into a radioactivity monitor (BarberColman Series 500). For measyrement of CO2 column 2 of the gas partitioner was by-passed. Alternately, CO2 was measured by injecting a gas sample into a sealed flask containing Hyamine hydroxide in the center well as described previously (9). Preparation of substrates. d,l-Tetrahydrofolate by hydrogenation of d,l-folic acid over platinum Me& enetetrahydrofolate (5,10-l4 CH2-H41folate) and It C-formaldehyde (New England uclear Corp.) N5-14C-Methyltetrahydrofolate (5- 18 CH3-H4-folate) of 5,10-14CH2-H4-folate with sodium borohydride
(d,l-H -folate was ~4.~10 . frEyred oxide ft 10). was prepared from He-folate (10 mCi per mmole) ft11). was prepared by reduction (12).
Unlabeled methylcobalamin (methyl-Co-5,6-dimethyl 'l~~~~~~ro,t,c~",~~~~e~~as prepared by the procedure of Miiller and Miiller (13). rsion of cyanocobalamin to hydroxocobalamin (14) and ;-;;g ",;i;h;i;F'z C-methyl iodide (10 mc per mmole) (15). All prepared substrates were stored at -20" in an inert atmosphere in sealed glass ampoules and shielded from light. RESULTS AND DISCUSSION: bacterial
cells
lost
the cells
when
phase not
are
of CH4 production
shown in Table
1.
were
by pressure
from Hz to C02:H2
by disrupted
Rates
cells.
disrupted (20:80) Disrupted
by stored,
Approximately
stimulated cells
125
frozen,
mixed
50% of the activity
and sonication. CH4 production
was
Changing by intact
made no CH4 in the absence
rumen
the gas
cells of Hz.
but
Vol. 79, No. 1, 1977
Table
BIOCHEMICAL
1.
AND BIOPHYSICAL
Rates of Methane Production Rumen Bacterial Cells.
Intact
Gas Phase
by Intact
Cells
H2:C02 (80:20) co2
I*
co2
Intact cells (0.5 ml cell fractions were tions continued for slopes following the to the system. II
I is is
the initial the final
The intact
bacterial
permitted
a low rate
continue
after
cells
02 dissolved is
known
cells
the Ribi
press
entirely
in
E. mobilis disruption.
42
0
(Fig.
1).
(Table
umole 1).
in
the first
h) which
CH4 production
was observed
This
the
was probably sensitivity
of CH4 synthesis
CH4 producers, bound
did
not
findings
of activity
126
1).
traces
(Fig.
(16,
40% after
disruption
1).
17)
resided
shows
ruminantium and pressure
that
in
after
This
in g.
of
bacteria
remained
by sonication reports
intact
The activity
that
enzyme(s)
solubilized
previous
of minute
pressure
(Fig.
The supematant
the CH4 producing
explain
with
by approximately
high
3 min sonication
g was devoid
both
to 02 of CH4 producing
more after
and not
result
was decreased
slightly
fraction.
at 15,000
These
some H2 (0.6
The high
the particulate
are membrane
(30 min to 2 h);
in CH4 production
and an additional
in other
98
of cell suspension before disruption) and Incubaincubated as described in the text. 6 h and rates were calculated from the linear initial 30-60 min lag caused by "oxygen shock"
lag
and decreased
min centrifugation unlike
271
produced
min)
The rate
3 min sonication
108
of H2 synthesis.
in buffers.
(1).
237
of CH4 production
(O-60
and disrupted
CH4/mg protein/h)
rate of CH4 production rate (2 h to 6 h).
cessation
An initial
Cells
27
II
*
and Disrupted
Disrupted
(millimicromoles H2
RESEARCH COMMUNICATIONS
cell-free
a 30 that, and
BIOCHEMICAL
Vol. 79, No. 1, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
640 600 560
120 80 40 0
30
60
90
120 TIME
150
180
210
240
(min)
Fig. 1. Bacterial cells (intact and disrupted) were incubated in an H2 atmosphere as described in text. Reaction mixtures contained 1 pcurie KH14C03 per flask and were started with protein addition (12.5 mg protein per flask). 14CH4 synthesis by intact cells, r-0; after 3 min sonication, Ur; after Ribi cell disruption and an additional 3 min sonication, o--.-n; after 15,000 g centrifugation, &---A.
extracts
(22,000
The 15,000
g supernatant)
g pellet
contained
In the reduction reduction and less study,
of CO2 to CH20 (18). than
1 mole
C02, in fact,
Earlier (100,000
almost
the addition
to the particulate
in this
did
required,
presumably
however,
require
is
laboratory
Fractionation
127
of this
in the
initia
stoichiometric
(3).
In the present of ATP to form
inhibitory.
had indicated stimulated
not
the addition
of ATP was slightly
of rumen bacteria
fraction.
not
activity.
activity.
used per mole of CH4 formed
fraction
experiments
g) supernatant
The requirement
of ATP is
show no methanogenic
allthemethanogenic
of CO2 to CH4 some ATP is
the rumen bacterial
CH4 from
of -M. ruminantium
that
high
CH4 production supernatant
speed when added
fraction
showed
Vol. 79, No. 1, 1977
Table
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
14 14 CH4 Synthesis from CO2 or 14CH30H by Intact Cell-free Particles of Rumen Bacteria.
2.
and
Substrate
Added to Incubation
14
d4c03 (cpm/mg
Intact
Cells
CH30H
protein/hr)
Cells
Expt.
I
121,600
36,000
Expt.
II
162,600
28,200
Cell-free
Particles
Expt.
I
123,915
37,917
Expt.
II
27,400
4,100
To the incubation mixture described in the text, was added 1 uCi IZH14C03 Endogenous CO2 in whole (10 mCi/mmole) or 1 uCi 14CH30H (3.3 mCi/mmole). cells was approx. 5 mM and in cell-free particles approx. 2 mM. In Expt. particles had I cell-free particles were used fresh; in Expt. II cell-free been stored frozen.
that
the stimulatory
be completely
replaced
g. barkerii retention
is
can grow
g. M.o.H.
not
addition
in the
and produce
atoms and without
a good substrate
for
and by cell-free
other
particles
particles
In cell-free did
not
suggests
decrease that
when stored,
lose
of rumen bacteria
the rate
of 14C0 2 reduction
128
with
producing
bacteria
than
the addition to l4 CH4 (data
including
to 14 CH4 both rate
indicated
endogenous (Table
Methanol,
CO2 (19).
at a lower
greater
and CO2 may not be reduced
rumen bacteria.
mixtures.
CH4 from CH30H with
2) but
with
and could
incubation
was converted
some activity
extracts
methanol
methane
(Table
of CO2 reduction to CH4 was probably 14 radioactivity of the CO2 undergoes dilution
fraction
equilibration
14 C-methanol
The rate
Cell-free
flavin
of FAD to the
in 1% methanol
In rumen bacteria,
(1).
cells
was entirely
by the
of the hydrogen
however,
intact
activity
by 14co
than since
CO2 of cell
the extracts.
2). of up to 30 mM methanol not
shown).
2'
This
to CH4 by a common pathway
in
BIOCHEMICAL
Vol. 79, No. 1, 1977
Table
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
14
14 CH4 and CO2 Synthesis by Cell-Free N5,-N10-14C-Methylenetetrahydrofolate, and 14C-Methylcobalamin.
3.
Particulate Fraction from N5-14C-Methyltetrahydrofolate
Rates of production (cpmjmg protein/h)
Substrate 14CH 4
14co 2 (initial
5,10- 14 CH2-H4-folate
1,740
5,10- 14 CH2-H4-folate plus
403
14C0 2 30 min)
(0.5
7,060
2,310
3,070
1,840
HCO; (50 mM)
5- 14 CH3-H4-folate
1,070
14 CH3-B12
4,440
610
--
60
--
To the incubation mixture described in the text were added: radioactive (0.5 pmole, 5 x lo5 dpm); ATP (10 mM); MgS04 (5 mM). Reaction mixtures incubated for 6 h in the dark and rates measured as in Table 1.
It
has been
c0nverte.d
cell-free
CH2-H4-folate mediates
by a decreased
it
Formate
which
impossible
is
to state
first
undergo
possibility
that
H4-folate
diluted
fraction (Table
high
to CH4 is
oxidized
rate
in g. if
(12).
equilibrate
by the observation
of HCO; to the incubation
129
with
12).
these after
C02, 5-CH3-H4-folate
inter30 min
and 5,10-CH2-
and formyl-tetrahydrofolate
results
groups
reduction with that
mixture
(ferredoxin
in Table
3,
of the H4-folate to CH4.
CO2 before 14
to
dehydrogenase
From the
and methylene
to CO2 before
(4,
CH4 from 5,10-
followed
to CO2 by for-mate
the methyl
derivatives
synthesized
14 CO2 formation
methyl-
M.o.H.
and CH -B 3 12 are
At the same time,
To yield
through
oxidation
supported
by the addition
of
substrate were
and --M. M.o.H.
also
3).
may be oxidized
present
derivatives
reduction
particulate
are
5-CH3-H4-folate
of -M. barkerii
of 14 CO2 release.
rate
formate.
is
extracts
was an initial
presumably
dependent)
5,10-CH2-H4-folate,
and 5-CH3-H4-folate
there
H4-folate
shown that
to CH4 by cell-free
Rumen bacterial
yield
to 3h)
The undergoing
CH4 radioactivity (Table
3).
is Although
Vol. 79, No. 1, 1977
BIOCHEMICAL
AND BIOPHYSICAL
I 2
I 1 TIME
Fig. 2. CH4 release from CH3-B12 by cell-free The reaction mixture contained: potassium 10 mM; ATP, 10 mM; CH3B12,~ q, 0.1 mM; *-*, 3 mM; and were incubated under H2 represents the same incubation mixture with
the conversion particulate
of the H4-folate fraction,
CO2 by the 15,000 No appreciable light
containing
reaction takes
totally
place
was formed
by soluble
bacteria.
There
emphasizes absence
from
mixtures (Fig. extracts
extract (12 mg) of C. pasteurianum. phosphate, (pH 7.0), 0.1 M; MgS04, -, 0.5 mM; -, 1mM; in room light. Lower line (U) 3 mM CH3-Bl2 incubated in the dark.
to CH4 was totally
and CH2-H4-folate fraction
CH4 was produced
to exclude
CH4 release
g supernatant
incubation
are incubated 2).
were
of rumen bacteria
from methylcobalamin the
I 3
(h)
derivatives
CH3-H4-folate
RESEARCH COMMUNICATIONS
In this
spurious
CH4 release
from
130
oxidized
(unpublished
(Table
were
H2 in room light,
non-specific
more than a non-methane (Fig. out
CH3-B 12 may result.
to
data).
If CH3-B12
was no CH release in the absence of light 4 the fact that unless such experiments are carried
of light,
on the
3).
experiment,
of C. pasteurianum,
readily
when precautions
mixtures under
dependent
0.5 pmole producing 2). in the
This total
From these
taken
of CH4
BIOCHEMICAL
Vol. 79, No. 1, 1977
results
it
synthesis
does not
appear
To date, ruminantium
by Bl2
only
had not been
that
can synthesize
show that
ruminantium
and g. mobilis
the particulate
fraction. are
routinely
active
activity
because
excessively
extracts.
It
tightly
the
high known
from --M. M.o.H.
Therefore,
that
is
that
and&L
are
enzymes in
than
those
g centrifugation too,
step.
the methanogenic
ACKNOWLEDGEMENTS: Mr. Wm. Cantwell.
4) it
M.
entirely
(15,000
g)
and -M. barkerii
is possible
that
the
has been overlooked
in the preparation
of these
cells
difficult
whereas
easily
are
disrupted
to break by sonication
the rumen bacteria
in _M. barkerii be ruled
is
associated
(1).
may be much more
or -M. barkerii.
cannot
activity
include
resides
from --M. M.o.H. (3,
of -M.
been used
in -M. -M.o.H.
It
which
of rumen bacteria
-M. ruminantium
transfer
of the present
in the supematant
g supernatant
may have
group
extracts
the activity
extracts
CO2 to CH4 has not been purified
reduces 30,000
bound
but
in CH4
form.
cell-free
and not cell
barkerii
methyl
of rumen bacteria
extracts
g forces
the methanogenic membrane
organisms
in cell-free
an intermediate
The results
CH4
fraction
30,000
bound
17).
extracts
is
bacteria,
to prepare
do synthesize
in the
these
in the protein
methanogenic
isolated
in
CH4 (16,
cell-free
RESEARCH COMMUNICATIONS
methylcobalamin
possible
or membrane
Since
methanogenic
cells
when
it
investigation
free
Possibly,
by rumen bacteria.
can be catalyzed
in
that
AND BIOPHYSICAL
out with
The enzyme system or _M. M.o.H.
therefore,
that
the membrane
The authors acknowledge the expert technical This is Contribution No. 720 from the Animal
beyond in
these
fraction.
assistance of Research Institute.
REFERENCES: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Wolfe, R.S. (1971) Advances in Microbial Physiol. 5, 107-146. Stadtman, T.C. (1967) Annu. Rev. Microbial. 21, 121-146. Roberton, A.M. and Wolfe, R.S. (1969) Biochim. Biophys. Acta 192, 420-429. Blaylock, B.A. and Stadtman, T.C. (1966) Arch. Biochem. Biophys. 116, 138-152. Smith, P.H. and Hungate, R.E. (1958) J. Bacterial. 2, 713-718. Paynter, M.J.B. and Hungate, R.E. (1968) J. Bacterial. 95, 1943-1951. Bush, R.S. and Sauer, F.D. (1976) Biochem. J. 157, 325-331. Bush, R.S. and Sauer, F.D. (1977) .I. Biol. Chem. 252, 2657-2661. Sauer, F.D., Mahadevan, S. and Erfle, J.D. (1971) Biochim. Biophys. Acta 239, 26-32. Rabinowitz, J.C. and Pricer, W.E. (1957) J. Biol. Chem. 229, 321-328. Guest, J.R., Foster, M.A. and Woods, D.D. (1964) Biochem. J. 92, 488-496. Wood, J.M., Allan, A.M., Brill, W.J. and Wolfe, R.S. (1965) J. Biol. Chem. 240, 4564-4569. Muller, 0. and Miiller, G. (1962) Biochem. Zt. 336, 299-313.
131
Vol. 79, No. 1, 1977
14. 15. 16. 17. 18. 19.
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Kaczka, E., Wolf, D.E. and Folkers, K. (1949) J. Am. Chem. Sot. 71, 1154-1155. Johnson, A.W., Mervyn, L., Shaw, N. andsmith, E.L. (1963) J. Cheic Sot. 1912, 4146-4156. Bryant, M.P., McBride, B.C. and Wolfe, R.S. (1968) J. Bact. 95, 1118-1123. Tzeng, S.F., Bryant, M.P. and Wolfe, R.S. (1975) J. Bact. 121, 192-196. Thauer, R.K., Jungermann, K. and Decker, K. (1977) Bacteriological Reviews 41, 100-179. Stadtman, T.C. and Blaylock, B.A. (1966) Fed. Proc. 2, 1657-1661.
132
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
METHANE PRODUCTION BY CELL-FREE
PARTICULATE
FRACTION OF RUMEN BACTERIA
F.D. Sauer, R.S. Bush, S. Mahadevan and J.D. Erfle Animal Research Institute, Agriculture Canada, Ottawa, Ontario, Canada, KlA OC4 .
Received
September
21,197i'
retained methanogenic activity when stored at -60" SUMMARY: Rumen bacteria under H2. This activity, which resides in Methanobacterium ruminantium and Methanobacterium mobilis, is not lost when the cells are broken, as has been suggested. Unlike in Methanosarcina barkerii and Methanobacterium M.o.H., in rumen bacteria methanogenic enzymes are not soluble but readily precipitated at 15,000 g. Methane was synthesized from tetrahydrofolate derivatives but at slower rates than from C02. From the data, it was not possible to determine if methyland methylene tetrahydrofolate were oxidized to CO2 prior to reduction to CH4. In room light, CH3-Bl2 was reduced to CH4 non-enzymatically in the presence of protein. When the reactions were carried out in the dark, very little CH4 was formed from CH3-Bl2 by rumen bacterial enzymes. The cell-free particulate fraction did not require added ATP for methanogenesis but showed an absolute requirement for H2. INTRODUCTION: hydrogen
Most methane
and carbon
Methanosarcina (2).
are
oscillation at 30,000
can utilize
out with
prepared
and French
pressure
cell
ruminantium
(approx. (5,
and stored
fraction
after
production
for
the pathway
M.o.H.
and fi.
oscillation
(3)
disruption
(4)
(1).
with
In addition,
growth
and methane
of methane
formation Cell-free
barkerii.
or by combined
followed
sonic
by centrifugation
25 to 60 min.
production
report
with
requirements
and carbon
and acetate
Methanobacterium by sonic
flora
In this
dealing
nutritional
energy
methanol
prepared
Methanobacterium
methane
both
generally
g for
the rumen
have simple
supplying
Much of the work
has been carried extracts
dioxide
barkerii
production
bacteria
4 x lo8 6).
complete
of methane
cells
We have
as described
we show that
and Methanobacterium per ml)
observed
previously
methanogenic
cell
by the membrane
and together that
(7),
account
total
methane.
in the particulate
of intermediates
of rumen bacterial
for
in
pellets,
synthesize
is entirely
The role fraction
are present
rumen bacterial actively
activity
disruption.
mobilis
in the cells
was
examined.
Copyright All rights
0 1977
by Academic Press, Inc. in any form reserved.
of reproduction
124 ISSN
0006-291
X
Vol. 79, No. 1, 1977
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MATERIALS AND METHODS: Preparation of cell-free extract. Rumen bacterial pellets were prepared as described (7) but stored at -60" in clarified rumen fluid under HT. To thawed 10 ml pellets (ZOO to 250 mg protein) was added with continuous H; gassing: FAD (1 mM); CoA, (0.1 mM); pyruvate, (5 mM); and pyruvate lyase isolated from Clostridium pasteurianum (8) (' 2 mg). These additions kept the bacterial cell fractions fully reduced through subsequent steps provided that extreme care was used to exclude 02 and maintain a constant N2 or H2 atmosphere. Cells were disrupted by 3 min sonication in a Bronson sonifier cell disruptor (90 to 100 watts) in cycles of 15 s on and 45 s off. The partially disrupted cell suspension was then put through an R-l Ribi cell fractionator at 25,000 p.s.i. This was followed by an additional 3 min sonication as before. The cell extract was then centrifuged 30 min at 2380 g (unless specified otherwise) under H2 in tightly stoppered tubes. A one ml aliquot of this cell extract was centrifuged at 100,000 g. The resulting pellet was fixed in glutaraldehydejosmium tetraoxide and stained with uranyl acetate/lead citrate for examination in a Phillips 300 electron microscope. No intact bacterial cells were found. Incubation and assay procedure. Cell fractions (0.5 ml) were incubated at 38' in 0.1 M potassium phosphate buffer (pH 7.0) containing 1 mM Na2S.9H20 (final vol. 1.0 ml) in 5 ml Erlenmeyer flasks sealed with serum bottle rubber stoppers. Additions were made as specified in legends to Figures and Tables. Before addition of enzyme, each flask was evacuated and flushed with Hz for three 1 min periods. Substrate and enzyme were added by microsyringe to Hz filled flasks before incubation. Gas samples were withdrawn at timed intervals with a gas syringe (Precision Sampling Corp., Baton Rouge, La.) and assayed on a gas partitioner (Fisher Model 1200) coupled to a recording integrator (Infotronics CRS 1lOA). The effluent gas, after partitioning, was passed through copper tubing into a combustion furnace and the;t4into a radioactivity monitor (BarberColman Series 500). For measyrement of CO2 column 2 of the gas partitioner was by-passed. Alternately, CO2 was measured by injecting a gas sample into a sealed flask containing Hyamine hydroxide in the center well as described previously (9). Preparation of substrates. d,l-Tetrahydrofolate by hydrogenation of d,l-folic acid over platinum Me& enetetrahydrofolate (5,10-l4 CH2-H41folate) and It C-formaldehyde (New England uclear Corp.) N5-14C-Methyltetrahydrofolate (5- 18 CH3-H4-folate) of 5,10-14CH2-H4-folate with sodium borohydride
(d,l-H -folate was ~4.~10 . frEyred oxide ft 10). was prepared from He-folate (10 mCi per mmole) ft11). was prepared by reduction (12).
Unlabeled methylcobalamin (methyl-Co-5,6-dimethyl 'l~~~~~~ro,t,c~",~~~~e~~as prepared by the procedure of Miiller and Miiller (13). rsion of cyanocobalamin to hydroxocobalamin (14) and ;-;;g ",;i;h;i;F'z C-methyl iodide (10 mc per mmole) (15). All prepared substrates were stored at -20" in an inert atmosphere in sealed glass ampoules and shielded from light. RESULTS AND DISCUSSION: bacterial
cells
lost
the cells
when
phase not
are
of CH4 production
shown in Table
1.
were
by pressure
from Hz to C02:H2
by disrupted
Rates
cells.
disrupted (20:80) Disrupted
by stored,
Approximately
stimulated cells
125
frozen,
mixed
50% of the activity
and sonication. CH4 production
was
Changing by intact
made no CH4 in the absence
rumen
the gas
cells of Hz.
but
Vol. 79, No. 1, 1977
Table
BIOCHEMICAL
1.
AND BIOPHYSICAL
Rates of Methane Production Rumen Bacterial Cells.
Intact
Gas Phase
by Intact
Cells
H2:C02 (80:20) co2
I*
co2
Intact cells (0.5 ml cell fractions were tions continued for slopes following the to the system. II
I is is
the initial the final
The intact
bacterial
permitted
a low rate
continue
after
cells
02 dissolved is
known
cells
the Ribi
press
entirely
in
E. mobilis disruption.
42
0
(Fig.
1).
(Table
umole 1).
in
the first
h) which
CH4 production
was observed
This
the
was probably sensitivity
of CH4 synthesis
CH4 producers, bound
did
not
findings
of activity
126
1).
traces
(Fig.
(16,
40% after
disruption
1).
17)
resided
shows
ruminantium and pressure
that
in
after
This
in g.
of
bacteria
remained
by sonication reports
intact
The activity
that
enzyme(s)
solubilized
previous
of minute
pressure
(Fig.
The supematant
the CH4 producing
explain
with
by approximately
high
3 min sonication
g was devoid
both
to 02 of CH4 producing
more after
and not
result
was decreased
slightly
fraction.
at 15,000
These
some H2 (0.6
The high
the particulate
are membrane
(30 min to 2 h);
in CH4 production
and an additional
in other
98
of cell suspension before disruption) and Incubaincubated as described in the text. 6 h and rates were calculated from the linear initial 30-60 min lag caused by "oxygen shock"
lag
and decreased
min centrifugation unlike
271
produced
min)
The rate
3 min sonication
108
of H2 synthesis.
in buffers.
(1).
237
of CH4 production
(O-60
and disrupted
CH4/mg protein/h)
rate of CH4 production rate (2 h to 6 h).
cessation
An initial
Cells
27
II
*
and Disrupted
Disrupted
(millimicromoles H2
RESEARCH COMMUNICATIONS
cell-free
a 30 that, and
BIOCHEMICAL
Vol. 79, No. 1, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
640 600 560
120 80 40 0
30
60
90
120 TIME
150
180
210
240
(min)
Fig. 1. Bacterial cells (intact and disrupted) were incubated in an H2 atmosphere as described in text. Reaction mixtures contained 1 pcurie KH14C03 per flask and were started with protein addition (12.5 mg protein per flask). 14CH4 synthesis by intact cells, r-0; after 3 min sonication, Ur; after Ribi cell disruption and an additional 3 min sonication, o--.-n; after 15,000 g centrifugation, &---A.
extracts
(22,000
The 15,000
g supernatant)
g pellet
contained
In the reduction reduction and less study,
of CO2 to CH20 (18). than
1 mole
C02, in fact,
Earlier (100,000
almost
the addition
to the particulate
in this
did
required,
presumably
however,
require
is
laboratory
Fractionation
127
of this
in the
initia
stoichiometric
(3).
In the present of ATP to form
inhibitory.
had indicated stimulated
not
the addition
of ATP was slightly
of rumen bacteria
fraction.
not
activity.
activity.
used per mole of CH4 formed
fraction
experiments
g) supernatant
The requirement
of ATP is
show no methanogenic
allthemethanogenic
of CO2 to CH4 some ATP is
the rumen bacterial
CH4 from
of -M. ruminantium
that
high
CH4 production supernatant
speed when added
fraction
showed
Vol. 79, No. 1, 1977
Table
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
14 14 CH4 Synthesis from CO2 or 14CH30H by Intact Cell-free Particles of Rumen Bacteria.
2.
and
Substrate
Added to Incubation
14
d4c03 (cpm/mg
Intact
Cells
CH30H
protein/hr)
Cells
Expt.
I
121,600
36,000
Expt.
II
162,600
28,200
Cell-free
Particles
Expt.
I
123,915
37,917
Expt.
II
27,400
4,100
To the incubation mixture described in the text, was added 1 uCi IZH14C03 Endogenous CO2 in whole (10 mCi/mmole) or 1 uCi 14CH30H (3.3 mCi/mmole). cells was approx. 5 mM and in cell-free particles approx. 2 mM. In Expt. particles had I cell-free particles were used fresh; in Expt. II cell-free been stored frozen.
that
the stimulatory
be completely
replaced
g. barkerii retention
is
can grow
g. M.o.H.
not
addition
in the
and produce
atoms and without
a good substrate
for
and by cell-free
other
particles
particles
In cell-free did
not
suggests
decrease that
when stored,
lose
of rumen bacteria
the rate
of 14C0 2 reduction
128
with
producing
bacteria
than
the addition to l4 CH4 (data
including
to 14 CH4 both rate
indicated
endogenous (Table
Methanol,
CO2 (19).
at a lower
greater
and CO2 may not be reduced
rumen bacteria.
mixtures.
CH4 from CH30H with
2) but
with
and could
incubation
was converted
some activity
extracts
methanol
methane
(Table
of CO2 reduction to CH4 was probably 14 radioactivity of the CO2 undergoes dilution
fraction
equilibration
14 C-methanol
The rate
Cell-free
flavin
of FAD to the
in 1% methanol
In rumen bacteria,
(1).
cells
was entirely
by the
of the hydrogen
however,
intact
activity
by 14co
than since
CO2 of cell
the extracts.
2). of up to 30 mM methanol not
shown).
2'
This
to CH4 by a common pathway
in
BIOCHEMICAL
Vol. 79, No. 1, 1977
Table
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
14
14 CH4 and CO2 Synthesis by Cell-Free N5,-N10-14C-Methylenetetrahydrofolate, and 14C-Methylcobalamin.
3.
Particulate Fraction from N5-14C-Methyltetrahydrofolate
Rates of production (cpmjmg protein/h)
Substrate 14CH 4
14co 2 (initial
5,10- 14 CH2-H4-folate
1,740
5,10- 14 CH2-H4-folate plus
403
14C0 2 30 min)
(0.5
7,060
2,310
3,070
1,840
HCO; (50 mM)
5- 14 CH3-H4-folate
1,070
14 CH3-B12
4,440
610
--
60
--
To the incubation mixture described in the text were added: radioactive (0.5 pmole, 5 x lo5 dpm); ATP (10 mM); MgS04 (5 mM). Reaction mixtures incubated for 6 h in the dark and rates measured as in Table 1.
It
has been
c0nverte.d
cell-free
CH2-H4-folate mediates
by a decreased
it
Formate
which
impossible
is
to state
first
undergo
possibility
that
H4-folate
diluted
fraction (Table
high
to CH4 is
oxidized
rate
in g. if
(12).
equilibrate
by the observation
of HCO; to the incubation
129
with
12).
these after
C02, 5-CH3-H4-folate
inter30 min
and 5,10-CH2-
and formyl-tetrahydrofolate
results
groups
reduction with that
mixture
(ferredoxin
in Table
3,
of the H4-folate to CH4.
CO2 before 14
to
dehydrogenase
From the
and methylene
to CO2 before
(4,
CH4 from 5,10-
followed
to CO2 by for-mate
the methyl
derivatives
synthesized
14 CO2 formation
methyl-
M.o.H.
and CH -B 3 12 are
At the same time,
To yield
through
oxidation
supported
by the addition
of
substrate were
and --M. M.o.H.
also
3).
may be oxidized
present
derivatives
reduction
particulate
are
5-CH3-H4-folate
of -M. barkerii
of 14 CO2 release.
rate
formate.
is
extracts
was an initial
presumably
dependent)
5,10-CH2-H4-folate,
and 5-CH3-H4-folate
there
H4-folate
shown that
to CH4 by cell-free
Rumen bacterial
yield
to 3h)
The undergoing
CH4 radioactivity (Table
3).
is Although
Vol. 79, No. 1, 1977
BIOCHEMICAL
AND BIOPHYSICAL
I 2
I 1 TIME
Fig. 2. CH4 release from CH3-B12 by cell-free The reaction mixture contained: potassium 10 mM; ATP, 10 mM; CH3B12,~ q, 0.1 mM; *-*, 3 mM; and were incubated under H2 represents the same incubation mixture with
the conversion particulate
of the H4-folate fraction,
CO2 by the 15,000 No appreciable light
containing
reaction takes
totally
place
was formed
by soluble
bacteria.
There
emphasizes absence
from
mixtures (Fig. extracts
extract (12 mg) of C. pasteurianum. phosphate, (pH 7.0), 0.1 M; MgS04, -, 0.5 mM; -, 1mM; in room light. Lower line (U) 3 mM CH3-Bl2 incubated in the dark.
to CH4 was totally
and CH2-H4-folate fraction
CH4 was produced
to exclude
CH4 release
g supernatant
incubation
are incubated 2).
were
of rumen bacteria
from methylcobalamin the
I 3
(h)
derivatives
CH3-H4-folate
RESEARCH COMMUNICATIONS
In this
spurious
CH4 release
from
130
oxidized
(unpublished
(Table
were
H2 in room light,
non-specific
more than a non-methane (Fig. out
CH3-B 12 may result.
to
data).
If CH3-B12
was no CH release in the absence of light 4 the fact that unless such experiments are carried
of light,
on the
3).
experiment,
of C. pasteurianum,
readily
when precautions
mixtures under
dependent
0.5 pmole producing 2). in the
This total
From these
taken
of CH4
BIOCHEMICAL
Vol. 79, No. 1, 1977
results
it
synthesis
does not
appear
To date, ruminantium
by Bl2
only
had not been
that
can synthesize
show that
ruminantium
and g. mobilis
the particulate
fraction. are
routinely
active
activity
because
excessively
extracts.
It
tightly
the
high known
from --M. M.o.H.
Therefore,
that
is
that
and&L
are
enzymes in
than
those
g centrifugation too,
step.
the methanogenic
ACKNOWLEDGEMENTS: Mr. Wm. Cantwell.
4) it
M.
entirely
(15,000
g)
and -M. barkerii
is possible
that
the
has been overlooked
in the preparation
of these
cells
difficult
whereas
easily
are
disrupted
to break by sonication
the rumen bacteria
in _M. barkerii be ruled
is
associated
(1).
may be much more
or -M. barkerii.
cannot
activity
include
resides
from --M. M.o.H. (3,
of -M.
been used
in -M. -M.o.H.
It
which
of rumen bacteria
-M. ruminantium
transfer
of the present
in the supematant
g supernatant
may have
group
extracts
the activity
extracts
CO2 to CH4 has not been purified
reduces 30,000
bound
but
in CH4
form.
cell-free
and not cell
barkerii
methyl
of rumen bacteria
extracts
g forces
the methanogenic membrane
organisms
in cell-free
an intermediate
The results
CH4
fraction
30,000
bound
17).
extracts
is
bacteria,
to prepare
do synthesize
in the
these
in the protein
methanogenic
isolated
in
CH4 (16,
cell-free
RESEARCH COMMUNICATIONS
methylcobalamin
possible
or membrane
Since
methanogenic
cells
when
it
investigation
free
Possibly,
by rumen bacteria.
can be catalyzed
in
that
AND BIOPHYSICAL
out with
The enzyme system or _M. M.o.H.
therefore,
that
the membrane
The authors acknowledge the expert technical This is Contribution No. 720 from the Animal
beyond in
these
fraction.
assistance of Research Institute.
REFERENCES: 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
Wolfe, R.S. (1971) Advances in Microbial Physiol. 5, 107-146. Stadtman, T.C. (1967) Annu. Rev. Microbial. 21, 121-146. Roberton, A.M. and Wolfe, R.S. (1969) Biochim. Biophys. Acta 192, 420-429. Blaylock, B.A. and Stadtman, T.C. (1966) Arch. Biochem. Biophys. 116, 138-152. Smith, P.H. and Hungate, R.E. (1958) J. Bacterial. 2, 713-718. Paynter, M.J.B. and Hungate, R.E. (1968) J. Bacterial. 95, 1943-1951. Bush, R.S. and Sauer, F.D. (1976) Biochem. J. 157, 325-331. Bush, R.S. and Sauer, F.D. (1977) .I. Biol. Chem. 252, 2657-2661. Sauer, F.D., Mahadevan, S. and Erfle, J.D. (1971) Biochim. Biophys. Acta 239, 26-32. Rabinowitz, J.C. and Pricer, W.E. (1957) J. Biol. Chem. 229, 321-328. Guest, J.R., Foster, M.A. and Woods, D.D. (1964) Biochem. J. 92, 488-496. Wood, J.M., Allan, A.M., Brill, W.J. and Wolfe, R.S. (1965) J. Biol. Chem. 240, 4564-4569. Muller, 0. and Miiller, G. (1962) Biochem. Zt. 336, 299-313.
131
Vol. 79, No. 1, 1977
14. 15. 16. 17. 18. 19.
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Kaczka, E., Wolf, D.E. and Folkers, K. (1949) J. Am. Chem. Sot. 71, 1154-1155. Johnson, A.W., Mervyn, L., Shaw, N. andsmith, E.L. (1963) J. Cheic Sot. 1912, 4146-4156. Bryant, M.P., McBride, B.C. and Wolfe, R.S. (1968) J. Bact. 95, 1118-1123. Tzeng, S.F., Bryant, M.P. and Wolfe, R.S. (1975) J. Bact. 121, 192-196. Thauer, R.K., Jungermann, K. and Decker, K. (1977) Bacteriological Reviews 41, 100-179. Stadtman, T.C. and Blaylock, B.A. (1966) Fed. Proc. 2, 1657-1661.
132
