Methanogenesis from acetate: a comparison of the acetate metabolism in<i>Methanothrix soehngenii</i>and<i>Methanosarcina</i>spp.

Abstract

Acetate is the precursor of approximately two-thirds of the methane produced in anaerobic bioreactors. Only two genera of methanogenic archae are known to use acetate as sole energy source: &lt;it&gt;Methanosarcina&lt;/it&gt; and &lt;it&gt;Methanothrix. Methanosarcina&lt;/it&gt; appears to be a generalist with a high growth rate, but low affinity for acetate. &lt;it&gt;Methanothrix&lt;/it&gt; is a specialist having a high affinity for acetate, but low growth rate. &lt;it&gt;Methanothrix&lt;/it&gt; shows a much lower minimum threshold for acetate utilization (7–70 μM) than &lt;it&gt;Methanosarcina&lt;/it&gt; (0.2–1.2 mM). This is consistent with the evidence that &lt;it&gt;Methanothrix&lt;/it&gt; is found in environments with low acetate concentrations. The acetate degradation by acetotrophic methanogens starts with an activation of acetate to acetyl-coenzyme A. In &lt;it&gt;Methanosarcina&lt;/it&gt; spp. this activation is catalysed by an acetate kinase/phosphotransacetylase system at the expense of one ATP. Acetyl-coenzyme A synthetase activates acetate in &lt;it&gt;Methanothrix&lt;/it&gt;, with concomitant hydrolysis of one ATP to AMP and PP&lt;inf&gt;i&lt;/inf&gt;. Both enzyme systems have been purified and comparison of the kinetic properties confirmed the hypothesis that low acetate concentrations favour &lt;it&gt;Methanothrix&lt;/it&gt;. The gene encoding for acetyl-CoA synthetase of &lt;it&gt;Methanothrix&lt;/it&gt; was isolated from a genomic library and actively expressed in &lt;it&gt;Escherichia coli&lt;/it&gt;. The deduced amino acid sequence showed homology to proteins with similar function and contained two putative ATP binding sites. The most characteristic and complex enzyme involved in the acetate degradation by acetotrophic methanogens is carbon monoxide dehydrogenase. The enzyme has been purified from both &lt;it&gt;Methanothrix&lt;/it&gt; and &lt;it&gt;Methanosarcina&lt;/it&gt;, and represents 5–10% of the soluble protein of these microorganisms. CO dehydrogenase is proposed to catalyse both the cleavage of acetyl-CoA in a methyl-, carbonyl- and CoA-moiety, and the oxidation of the carbonyl group to CO&lt;inf&gt;2&lt;/inf&gt;. This multifunctional redox enzyme contains several iron, acid-labile sulfur and nickel atoms. These atoms are arranged into several paramagnetic complexes, which have been studied by EPR spectroscopy. The low spin recovery of the different paramagnetic centers makes statements about structure and functions difficult. There are good spectroscopic and genetic indications that the CO dehydrogenase of &lt;it&gt;Methanothrix&lt;/it&gt; contains at least one ferrodoxin-like [4Fe-4S] cluster, which could play a role in the electron transfer of the CO oxidation. Further, in EPR spectra of concentrated samples of CO dehydrogenase from &lt;it&gt;Methanothrix&lt;/it&gt; a very unusual signal was observed, which showed great similarity to putative [6Fe-6S] prismane clusters. The final step in the methanogenesis from acetate, the reduction of methyl-coenzyme M, is catalysed by methyl-coenzyme M methylreductase. The enzyme purified from &lt;it&gt;Methanothrix&lt;/it&gt; and &lt;it&gt;Methanosarcina&lt;/it&gt; showed great homology with the methyl-CoM methylreductase of other methanogenic archae, although the specific activity was rather low (60–125 nmol min−1 mg−1). The reduction of the heterodisulfide between coenzyme M and component B is proposed to be the common site for energy conservation in all methanogens. Acetoclastic methanogens, however, need additional sites of energy conservation to compensate for their high energy input in acetate activation. The oxidation of CO to CO&lt;inf&gt;2&lt;/inf&gt; could form one possible site. The partially membrane-associated pyrophosphatase of &lt;it&gt;Methanothrix&lt;/it&gt; could form another site of energy conservation.