Vol. 58, No. 10
Carbon Monoxide, Hydrogen, and Formate Metabolism
during Methanogenesis from Acetate by Thermophilic
Cultures of Methanosarcina and Methanothrix Strains
S. H. ZINDER* AND T. ANGUISH
Section ofMicrobiology, Wing Hall, Cornell University, Ithaca, New York 14853
Received 18 May 1992/Accepted 22 July 1992
CO and H2 have been implicated in methanogenesis from acetate, but it is unclear whether they are directly
involved in methanogenesis or electron transfer in acetotrophic methanogens. We compared metabolism ofH2,
CO, and formate by cultures of the thermophilic acetotrophic methanogens Methanosarcina thermophila TM-1
and Methanothrix sp. strain CALS-1. M. thermophila accumulated H2 to partial pressures of 40 to 70 Pa (1 Pa
= 0.987 x 10-5 atm), as has been previouslyreported for this and otherMethanosarcina cultures. In contrast,
Methanothrix sp. strain CALS-1 accumulated H2 to maximum partial pressures near 1 Pa. Growing cultures
of Methanothrix sp. strain CALS-1 initially accumulated CO, which reached partial pressures near 0.6 Pa
(some CO came from the rubber stopper) during the middle ofmethanogenesis; this was followed by a decrease
in CO partial pressures to less than 0.01 Pa by the end ofmethanogenesis. Accumulation or consumption ofCO
by cultures of M. thermophila growing on acetate was not detected. Late-exponential-phase cultures of
Methanothrix sp. strain CALS-1, in which the CO partial pressure was decreased by flushing with N2-CO2,
accumulated CO to 0.16 Pa, whereas cultures to which ca. 0.5 Pa of CO was added consumed CO until it
reached this partial pressure. Cyanide (1 mM) blocked CO consumption but not production. High partial
pressures of H2 (40 kPa) inhibited methanogenesis from acetate by M. thermophila but not by Methanothrir sp.
strain CALS-1, and 2 kPa of CO was not inhibitory to M. thermophila but was inhibitory to Methanothrix sp.
strain CALS-1. Levels of CO dehydrogenase, hydrogenase, and formate dehydrogenase in Methanothrix sp.
strain CALS-1 were 9.1, 0.045, and 5.8pmolofviologen reduced min
that CO plays a role in Methanothrix sp. strain CALS-1 similar to that of H2 inM. thermophila and are consistent
with the conclusion that CO is an intermediate in a catabolic or anabolic pathway in Methanothrix sp. strain
CALS-1; however, they could also be explained by passive equilibration of CO with a metabolic intermediate.
mg ofprotein-. These results suggest
There are two genera ofmethanogens known to decarbox-
ylate acetate to CH4 and CO2- Methanosarcina and Metha-
nothrix (also called Methanosaeta ). Methanosarcina
cultures usually grow more rapidly on acetate than do
Methanothrix cultures; however, Methanothrix species are
capable of using acetate at lower concentrations, since they
have lower apparent Km values (13, 28, 30) and minimum
thresholds (10, 16, 25, 37) for acetate utilization. These
differences suggest that there are significant differences
between the pathways for acetate catabolism in these two
genera. One difference in metabolic pathways which may
partially explain this difference in acetate affinity is that
Methanosarcina species apparently use an acetate kinase-
phosphotransacetylase enzyme system to activate acetate to
acetyl coenzyme A (acetyl-CoA), whereas Methanothrix
species have high levels of acetyl-CoA synthetase, which
has a higher affinity for acetate than does acetate kinase but
uses an extra mole ofATP per mole of acetate activated (16).
Considerable circumstantial evidence has accrued indicat-
ing that H2 is involved in acetate catabolism in Methano-
sarcina species. Cultures of Methanosarcina thermophila
TM-1 produce H2 during methanogenesis from acetate (1,
23) until it reaches an equilibrium level near 50 Pa (ca. 5 x
10-4 atm [1 atm = 1.01 x 105 Pa]). When the H2 partial
pressure was decreased by flushing the culture headspace of
M. thermophila or increased slightly by adding H2, it re-
turned to near 50 Pa within a few hours (1, 23) in cultures
that were actively catabolizing acetate. H2 production during
methanogenesis from acetate has also been found in meso-
philic Methanosarcina cultures (3, 20, 23). Furthermore, H2
or another reductant is required for methanogenesis from
acetate by Methanosarcina cell extracts (8, 21) and H2 is
also produced stoichiometrically from acetyl-CoA by those
extracts (8). Methanogenesis from acetate is inhibited by H2
in many but not all Methanosarcina strains (8). Inhibition of
methanogenesis from acetate by H2 in M. thermophila was
first reported by Mah et al. (24) and has been recently
described in more detailed studies by Ahring et al. (1). The
mechanism of inhibition is not understood, but H2 may
unbalance electron flow, so that fermentation of acetate
cannot proceed. This evidence is consistent with a model in
which hydrogen cycling plays a role in acetate metabolism
similar to that proposed for sulfate-reducing bacteria (27),
although such a model is far from proven for acetate catab-
olism by Methanosarcina species.
There is also evidence linking CO metabolism and meth-
anogenesis from acetate. An enzyme with carbon monoxide
dehydrogenase (CODH; carbon monoxide:methyl viologen
oxidoreductase) activity is a key enzyme in the pathwayin
methanogenesis from acetate (35) and has been shown to
disassemble acetyl-CoA into a methyl group, a carbonyl
group, and CoA (11). CODH activity is high in both Meth-
anosarcina and Methanothrix species (11, 15, 19, 33). In
some circumstances, free CO can equilibrate with an en-
zyme-bound carbonyl intermediate, but it is not clear that
in the pathway
converted to H2 and CO2 by mesophilic acetate-grown
is directly involved
(8). CO was
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1992,p.3323-3329
Copyright © 1992, American Society for Microbiology
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VOL. 58, 1992