JOURNAL OF BACTERIOLOGY, Feb. 1980, p. 694-9 Vol. 141, No.

2
0021-9193/80/02-0694/05$02.00/0

Origin of Hydrogen in Methane Produced by

Methanobacterium thermoautotrophicum
LACY DANIELS,t GAIL FULTON, ROBIN W. SPENCER,t AND WILLIAM H. ORME-JOHNSONt*
Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706

The production of deuterated methane by Methanobacterium thermoauto-

trophicum in H20-D20 mixtures was examined by high-resolution mass spectrom-
etry. The hydrogen in the methane arose solely from water and not from hydrogen
gas. Hydrogen gas served only as an electron source in methanogenesis. A whole-
cell product isotope discrimination of 1.5 favoring hydrogen over deuterium was
observed in methane production in 81 atom% deuterated water. The distribution
of deuterated methane species is described by a simple model of the overall
reaction.
The bacterium Methanobacterium thermo- from water. More recently, Sauer et al. (19) have
autotrophicum uses carbon dioxide as both sole suggested that hydrogen gas rather than water
carbon source and catabolic electron acceptor. is the source of hydrogen in methane produced
Some 95% of its total carbon flux is catabolic in by M. ruminantium based on experiments in
the formal reaction of equation 1, the net reduc- which 3H2 and 3H20 were employed. Fuchs et al.
tion of carbon dioxide to methane by four equiv- (10) have measured the deuterium enrichment
alents of hydrogen gas (23, 25): of M. thermoautotrophicum total cell material
4H2 + CO2 -*CH4 + 2H20 (1) compared with natural abundance in water and
hydrogen gas and concluded that most cellular
Although this carbon must pass through the hydrogen is derived from water, deuterium en-
oxidation states equivalent to formate, formal- richment in methane was not measured.
dehyde, and methanol, only the methanol level The reactions comprising equation 1 are likely
intermediate has been identified, as methyl co- to begin with one or more hydrogenases which,
enzyme M, by Wolfe and colleagues (14, 22, 25). by analogy to the hydrogenases in other genera,
To begin our studies of the sequence of enzy- (18) are likely to catalyze the exchange to H2
matic reactions that must comprise equation 1, and D20 to produce HD and D2 that we report
we wish to establish the origin of the hydrogen here. If a hydrogenase catalyzed the heterolytic
in the product methane as either water or hy- scission of hydrogen gas to a proton and enzyme-
drogen gas, or both. ("Protium" and "deute- bound hydride (11, 13, 18) and then transferred
rium" and H and D are used to refer to specific the hydride directly to a nonexchangeable redox
isotopes of hydrogen; "hydrogen" refers to H or coenzyme such as the 8-hydroxy-5-deazaflavin
D regardless of molecular attachment, "hydro- factor 420 (F420) (2, 9, 26) or nicotinamides, it is
gen gas" and "water" refer to H2, HD, or D2, and reasonable that that hydrogen might be again
H20 or D20 and any mixtures thereof.) Both transferred directly in one or more of the reduc-
origins have been proposed. Pine and Barker tive steps of methanogenesis.
(16) reported (no data shown) that a mixed We have addressed these questions with high-
culture grown on ethanol in D20 produced CD4. resolution mass spectroscopy of the methane
Pine and Vishniac (17) showed that enrichment and hydrogen gas species formed during meth-
cultures from San Francisco Bay mud produced ane production by M. thermoautotrophicum
CHD3 from CD3COOH, suggesting that one hy- from H2 and C02 in D20. We found that the
drogen on acetate-derived methane comes from hydrogens in methane clearly arise ultimately
water. Neither of the above experiments ex- from water and not hydrogen gas. A model pre-
cludes direct hydrogen incorporation from hy- dicting the distribution of deuterated methane
drogen gas. Penley and Wood (15) have shown species as a function of the isotope discrimina-
that in methane production from methylcobal- tion and deuterium enrichment in water is pre-
amin by extracts of M. bryantii (4), the hydro- sented.
gens on the methyl ligand of methylcobalamin
are retained and the fourth hydrogen comes MATERIALS AND METHODS
t Present address: Department of Chemistry, Massachu- Chemicals and gases. All chemicals used were of
setts Institute of Technology, Cambridge, MA 02139. reagent grade. Matheson Scientific, Inc. supplied H2-
694
VOL. 141, 1980 HYDROGEN IN M. THERMOAUTOTROPHICUM METHANE
C02 (80:20, vol/vol, premixed) and prepurified N2.
Deuterium oxide (99.7 atom% D) was purchased from
Aldrich.
Growth of cells. M. thermoautotrophicum strain
AH (27), kindly provided by J. G. Zeikus, was culti-
vated at 600C as described previously (6) by the tech-
nique of Balch and Wolfe (5) with pressurized 60-ml
Wheaton serum vials.
Determination of methane. Total methane in
cultures and experiments was determined with a
model 9500 gas chromatograph (Carle Instruments)
with a 1-m Porapak R aluminum column at 88°C,
nitrogen as the carrier gas, and a flame ionization
detector. Gas samples of 0.3 ml were removed from
vials and injected with a 1-ml Glas Pac syringe with
Teflon pressure lock device as described previously
(6).
Isotopic analysis. Deuterium enrichment in meth-
ane and hydrogen gas was determined with an AEI
MS-9 high-resolution mass spectrometer. Each sample
( to 6 ml) was introduced through a septum to the 2-
liter reservoir; the ionization potential was set at 70
eV. Resolution was greater than 4,000, which was
sufficient to resolve peaks at nominal mle 20 differing
in 0.005 mle. This resolution was necessary to separate
the methane species and their fragments from H20,
D20, and other background ions when small samples
were introduced. The distribution of methane species
was calculated by sequentially subtracting from the
high-mass end the parent ion and expected fragments
from the total observed mass spectrum. (A detailed
algorithm including the data from references [1] and
[8] is available from the authors on request.) The
fragmentation patterns of Diebler and Mohler (8) were
used; other reported values (1) indicate an inherent
variability in the fragmentation patterns even at the
same ionization potential. This variability is the main
factor limiting our accuracy in determining the distri-
bution of isotopic species of methane, the accuracy
decreasing for less deuterated methanes as errors ac-
cumulate upon repeated subtraction of fragment in-
tensities proportional to parent ion intensities.
The deuterium enrichment of water samples was
determined by proton nuclear magnetic resonance: the
concentration of H+ in the samples (at approximately
4.5 ppm from tetramethylsilane) was measured by
comparison to known concentrations of added dioxane
(at 3.55 ppm) in a Varian T-60 spectrometer.
Experiments. For the experiment of Fig. 1 and 2,
12 ml of a culture at mid-log phase (0.5 to 1 g [wet
weight] of cells per liter) was centrifuged anaerobically
in a 20-ml tube at 5,000 x g for 10 min, washed once
with 5 ml of anaerobic D20 containing 1 mM Na2S,
and resuspended in 5 ml of the same. The suspension
was transferred to a 60-ml serum-stoppered vial,
flushed, and then pressurized to 20 lb/in2 with H2-CO2
and incubated with vigorous shaking at 600C. Gas
samples for gas chromatography and mass spectral
analysis were removed at intervals with syringes
equipped with pressure lock devices.
For the experiment of Table 1, seven vials of 20 ml
of culture each at mid-log phase were centrifuged at
5,000 x g for 15 min under N2 and resuspended in a
total volume of 100 ml of buffer. This consisted of 98
ml of D20 (99.7 atom% D) and 2 ml of a concentrated
695
solution in water (containing per 100 ml of water:
KH2PO4, 1.8 g; NH4CI, 6.0 g; Na2CO3, 0.84 g; pH 6.8),
and was 2 mM in Na2S. This suspension was evacuated
and flushed with N2, and 30 ml was added to a 60-ml
vial under N2. The deuterium enrichment was de-
creased by the addition of 3 ml of H20. This vial was
flushed, pressurized, and incubated as before; after 4
h at 600C, samples were transferred to 12-ml evacu-
ated vials for later analysis.
Calculations and mathematical modeling. The
symbols used in this discussion are defined as follows:
n, atom fraction D in water; p, atom fraction H in total
methane; q, atom fraction D in total methane; a,
product isotope discrimination; XCH4, XCHD3, XCH2D2,
XCHD,, and XCD4, mole fraction of the subscript methane
species.
The distribution of product species in a reaction
that involves the acquisition of a proton or deuteron
from water is a function of both the inherent prefer-
ence of that reaction for H or D, called a, and the a
priori probability of acquiring H or D which is simply
measured by the enrichment of the H20-D20 mixture,
n. For a simple reaction, a is the ratio of reaction rates
in 100% H20 and 100% D20. In H20-D20 mixtures the
product distribution is given by the following: p =
mole fraction protio product = a/[a + (n/1 -n)];
q = mole fraction deutero product = 1 - p =
1/[a(l/n - 1) + 1]. In the production of methane from
C02, which involves four formal hydrogen transfers, if
all four transfers have the same a the final distribution
of methane species is given by (p + q)4, or XCH4 = p4;
XCH3D = 4p3q; XCH2D2 = 6p q ; XCHD, = 4pq3; XCD4 = q4.
The atom fraction H in total methane is given by
1/4(4XCH4 + 3XCH,D + 2XCH2D2 + XCHD3), and the atom
fraction D in total methane is given by 1/4(4XCH4 +
3XCHD3 + 2XCH2D2 + XCH3D). It is readily shown that
these atom fractions in total methane are identically
equal to p and q as defined above, respectively.
The same distribution of methane species, (p + q)4',
is given by a scheme in which there is no discrimina-
tion between H and D, that is a = 1, in any of the
reactions that place hydrogen on the methane carbon,
but there is a discrimination of a > 1 in the formation
of nonexchangeable hydrogen donors, e.g., dihydro-
F420 or NADPH, that are subsequently involved in
direct hydrogen transfer to the methane carbon.
RESULTS
Origin of hydrogen in methane. Cells
washed and resuspended in water (98.7 final
atom% D) produced methane after a lag of ap-
proximately 1 h, and methane production ceased
after 5 h (Fig. 1A). During this period, there was
exchange of deuterium from D20 into the H2 gas
phase, producing nearly equal amounts of HD
and D2 (Fig. 1B). Figure 2 shows that despite
the variation in the isotope content of the gas
phase, the deuterium enrichment in methane
was sufficiently high and invariant to prove that
all four hydrogens in methane have their ulti-
mate origins in water and not hydrogen gas. This
is particularly evident at the earlier time points.
696 DANIELS EI AL. J. BACTMOL.
T f I -r .
were not previously washed and centrifiged in
0.5 A- D20. Excessive manipulation of these extreme
anaerobes increases the lag time and decreases
0.44- A w, A the final methane production rates. The resus-
~c pension medium was buffered and contained
- ammonium salts. Deuterium enrichments in
E 0.3.t methane, water, and hydrogen gas were assayed
after 4 h of incubation.
c
qc Table 1 contains the reults of this experi-
U.)S 0.2 ment, from which the total methane deuterium
enrichment q is calculated. The product isotope
0.1
iOo. . .
0.0
100 . 0--
-.,, 0
' 80.
4..
80 60
S
C 16.
Co
ob60
0
a40 o
0
<~~~.
O - --

20.
C-

0 0.-- --I
0
0' O I 2 3 4 5
ho'urs
FIG. 2. Deutersum enrichment in methane, water,
o 1 .2 3 4 5
and hydrogen gas during methanogenesis by M. ther-
hours moautotrophicum. Symbols: 01 water; A, methane;
and 0, hydrogen gas. The errors associated with the
FIG. 1. Production of methane, HD, and D1 by M. measurement of hydrogengas and water enrichments
thermoautotrophicum in 98.7 atom% deuterated wa- falwithin the sbols. The waterenrichmentextrap-
ter. (A) Methane production as percent (vol/vol) of olation to zero time reflects the hydrogen gas ex-
gas phase. (B) Distribution of H2 (0), HD (A), and DA change reaction.
(@) as percent of total hydrogen gas.
TABLE 1. Distribution of deuterated methane
Deuterium isotope d mination in species from 81 atom%Jo deuterated medium
methaneeproductio. The difference in deu- Methane mole fraction
teium enrichment between methane and water Methane species
shown in Fig. 2 indicaes apreference for protons Observed Calculated
over deuterom in methanogene however, the CD4 0.26 0.29
enrr in quntitatively determining the isotope CHD3 0.43 0.42
effect was large at the high water enrichment CH2D2 0.30 0.23
usd and low levels of mee produced. Thus, CILD 0.01 0.05
a more favorable water enrichment of 81 atom% CH4 <0.01 0.005
D was used, and conditions were varied to max- "Final atom% were water, 81; hydrogen gas, 62;
D
imize production: more cels were used and methane, 73. Calclated methane mole fractions
than in the experiment of Fig. 2 and the cells are fron (p + q)4, with a - 1.5.
VOL. 141, 1980 HYDROGEN IN M. THERMOAUTOTROPHICUM METHANE
discrimination a = 1.5 ± 0.2 is then calculated
from q = 0.73 ± 0.02 and the atom fraction D in
water, n = 0.81 ± 0.01. With this value for a, the
model distribution of methane species, (p + q)4,
yields the predicted mole fraction of each species
also given in Table 1.
DISCUSSION
Since the hydrogens of methane originate in
water, equation 1 can be separated into distinct
oxidative and reductive halves:
4H2=8H++8e- (2)
8e- + 8 H+ + CO2- CH4 + 2 H20 (3)
Equation 2 catalyzed by one or more hydro-
is
genases, with the electrons initially reducing
such cofactors as F420, nicotinamides, flavins,
molybdenum cofactors, or iron-sulfur centers. In
D20, the reactions of equation 2 also describe
the exchange of H2 to HD and D2. Equation 3,
the reductive half of methanogenesis, represents
a sequence of reactions involving methyl coen-
zyme M and probably at least one other cur-
rently unknown carbon-carrying species (7, 14,
22, 25). In our experiments the H' produced in
equation 2 is diluted more than 50-fold by D+,
since the mole fraction of hydrogen in hydrogen
gas versus that in water is less than 0.02. Deu-
terated methane species could not arise from
direct transfer from HD or D2 produced via
equation 2, since the deuterium enrichment in
hydrogen gas is always much less than that in
water or methane, particularly at early times in
the experiment (Fig. 2). This essential measure-
ment has not been made in previous studies (16,
17, 19). The results of the experiment in 81
atom% deuterated water (Table 1) also support
this conclusion, since the deuterium enrichment
in methane is again significantly greater than
that of the hydrogen gas at the end of the
incubation.
Our conclusions corroborate those of Pine and
co-workers (16, 17) but are in sharp contrast to
those of Sauer et al. (19). Sauer et al. based their
conclusion that methane hydrogen arises from
hydrogen gas on the total radioactivity observed
in water, tritium gas, and methane, neglecting to
take into account the vast molar excess of hy-
drogen in water over that in hydrogen gas in
such an experiment. Using reasonable assump-
tions from their experimental description, the
specific activities of tritium in methane vs. water
and tritium gas support our conclusion that wa-
ter, not hydrogen gas, is the ultimate source of
the hydrogen in biogenic methane. (Assuming a
liquid volume of 1 ml and 5 ,umol of methane
produced, the following specific activities [in
microcuries per milligram-atom of H] were cal-
697
culated from Sauer et al. [19], experiment 2,
Table 2: 1,370 and 37 for the tracer 3H - H2 and
methane, respectively, and 1.0 and 0.22 for the
tracer 'H - H20 and methane, respectively. The
specific activity of methane relative to that of
the label source is clearly much greater when
water is the label source [0.22] than when hy-
drogen gas is the label source [0.03].)
Since reductions by reduced F420, like those of
its chemical relatives dihydro-5-deazaflavins
(21) and dihydronicotinamides (12), are likely to
involve direct and complete hydrogen transfer
from position 5, our conclusion is strongly sup-
ported by the recent observations of F. Jacobson
and C. Walsh (personal communication) that
tritium from tritium gas is not incorporated into
reduced F420 by partially purified H2:F420 oxido-
reductase from M. thermoautotrophicum AH
and by the references of Fuchs et al. (10) to
unpublished observations that hydrogenase
from M. thermoautotrophicum does not catalyze
a hydride transfer. It will be of interest to see
the extent to which the methane produced in
cell extracts is labeled after the addition of syn-
thetically deuterated or tritiated dihydro-F420,
NADH, and NADPH.
Our experiments were conducted only with M.
thermoautotrophicum, but the cohesiveness of
methanogens as a group (4, 23, 25), especially
with respect to their unique cofactors coenzyme
M and F420, suggests that the same source of
hydrogen will be found in all methanogens that
reduce C02 to methane. The same origin of
methane hydrogen is expected in formate-utiliz-
ing methanogens, since formate is likely to be
oxidized to C02 and H2 and this carbon dioxide
subsequently reduced to methane (24). How-
ever, when methanol, methylamine, or acetate
is used to produce methane by Methanosarcina
barkerii, the methyl hydrogens are probably
retained in the product methane and only a
single hydrogen is acquired from water (16, 17).
The apparent product isotope discrimination
of 1.5 calculated from the data of Table 1 is
larger than expected from an equilibrium isotope
effect (20); there are at least four ways in which
such an apparent isotope effect could arise in
this system. First, there may be true, large prod-
uct solvent isotope effects in one or more of the
steps in methanogenesis that attaches a nonex-
changeable hydrogen to the methanogenic car-
bon. Second, there may be a single true isotope
effect in the production of a hydride-transferring
cofactor such as dihydro-F420 or NADPH. Third,
the apparent effect may be due to a higher
protium concentration (i.e., lower water atom%
D) in intracellular water than in the extracellular
water (from which a is calculated). This could
arise from (i) a significant isotope effect in H+-
698 DANIELS ET AL.
D+ influx through the cell membrane, or (ii) a
significant internal production of H+ from H2
and hydrogenase inside the cell, or both. These
interesting and very different possibilities are
currently under investigation. The agreement
between the observed distribution of deuterated
methane species and that predicted by our sim-
ple model (Table 1) suggests that this problem
may not be prohibitively complex.
ACKNOWLEDGMENTS
L.D. is supported by a Public Health Service postdoctoral
fellowship from the National Institutes of Health; R.S. is a
fellow of the Helen Hay Whitney Foundation. This work was
supported by the College of Agriculture and Life Sciences,
University of Wisconsin, and by Public Health Service re-
search grant GM 17,170 from the National Institute of General
Medical Science.
We thank J. G. Zeikus for providing us with a culture of M.
thermoautotrophicum and Mel Micke for expert technical
assistance in obtaining the mass spectra, as well as C. Walsh
and F. Jacobson for permission to quote results in advance of
publication.
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