Methanogenesis or biomethanation is the formation of methane coupled to energy conservation by microbes known as methanogens. It is the fourth and final stage of anaerobic digestion. Organisms capable of producing methane for energy conservation have been identified only from the domain Archaea, a group phylogenetically distinct from both eukaryotes and bacteria, although many live in close association with anaerobic bacteria. The production of methane is an important and widespread form of microbial metabolism. In anoxic environments, it is the final step in the decomposition of biomass. Methanogenesis is responsible for significant amounts of natural gas accumulations, the remainder being thermogenic.

Methanogenesis in microbes is a form of anaerobic respiration. Methanogens do not use oxygen to respire; in fact, oxygen inhibits the growth of methanogens. The terminal electron acceptor in methanogenesis is not oxygen, but carbon. The two best described pathways involve the use of acetic acid (acetoclastic) or inorganic carbon dioxide (hydrogenotrophic) as terminal electron acceptors:

During anaerobic respiration of carbohydrates, H₂ and acetate are formed in a ratio of 2:1 or lower, so H₂ contributes only c. 33% to methanogenesis, with acetate contributing the greater proportion. In some circumstances, for instance in the rumen, where acetate is largely absorbed into the bloodstream of the host, the contribution of H₂ to methanogenesis is greater.

However, depending on pH and temperature, methanogenesis has been shown to use carbon from other small organic compounds, such as formic acid (formate), methanol, methylamines, tetramethylammonium, dimethyl sulfide, and methanethiol. The catabolism of the methyl compounds is mediated by methyl transferases to give methyl coenzyme M.

The biochemistry of methanogenesis involves the following coenzymes and cofactors: F420, coenzyme B, coenzyme M, methanofuran, and methanopterin.

The mechanism for the conversion of CH
₃–S bond into methane involves a ternary complex of the enzyme, with the substituents forming a structure α₂β₂γ₂. Within the complex, methyl coenzyme M and coenzyme B fit into a channel terminated by the axial site on nickel of the cofactor F430. One proposed mechanism invokes electron transfer from Ni(I) (to give Ni(II)), which initiates formation of CH
₄. Coupling of the coenzyme M thiyl radical (RS^.) with HS coenzyme B releases a proton and re-reduces Ni(II) by one electron, regenerating Ni(I).

Some organisms can oxidize methane, functionally reversing the process of methanogenesis, also referred to as the anaerobic oxidation of methane (AOM). Organisms performing AOM have been found in multiple marine and freshwater environments including methane seeps, hydrothermal vents, coastal sediments and sulfate-methane transition zones. These organisms may accomplish reverse methanogenesis using a nickel-containing protein similar to methyl-coenzyme M reductase used by methanogenic archaea. Reverse methanogenesis occurs according to the reaction:

Methanogenesis is the final step in the anaerobic decay of organic matter. During the decay process, electron acceptors (such as oxygen, ferric iron, sulfate, and nitrate) become depleted, while hydrogen (H₂) and carbon dioxide accumulate. Light organics produced by fermentation also accumulate. During advanced stages of organic decay, all electron acceptors become depleted except carbon dioxide. Carbon dioxide is a product of most catabolic processes, so it is not depleted like other potential electron acceptors.