Magrittrdigester reportThe three routes to methane in a digester, and the organisms that carry them
2026-09-26
A companion to What one digestate sample can tell you. A digester report names methanogens by genus and says which route to methane each one carries. This note explains the three routes, which organisms carry each, and what the literature says their presence tends to mean, so that the names in a report can be read rather than looked up.
Every litre of biomethane is made by archaea, and every methanogen finishes the job the same way: a methyl group bound to coenzyme M is reduced to methane by methyl-coenzyme M reductase, the enzyme encoded by mcrA (Thauer et al. 2008; Evans et al. 2019). What differs between them is what they feed into that last step. There are three substrate groups, and they give the three routes that a digester report names: carbon dioxide reduced with hydrogen (or formate), acetate, and methylated compounds such as methanol and methylamines (Liu and Whitman 2008).
mcrA is the marker a report uses to decide whether a genome is a methanogen at all. Anaerobic methane oxidisers carry it too and run the pathway in reverse, which is why the genus matters as much as the gene.
The routes are not interchangeable, and they are not carried by the same organisms. For sewage-sludge digesters at low ammonia, the textbook estimate is that about two thirds of the methane comes from acetate and most of the rest from hydrogen and carbon dioxide (Demirel and Scherer 2008). In ammonia-rich agricultural reactors the balance is often reversed, for reasons the section on acetate below explains. When a plant is under stress the organisms carrying each route change, and that is what DNA can see.
Hydrogenotrophic methanogens reduce CO2 to methane with hydrogen, and many can use formate instead. They are the most widespread methanogens and span several orders: Methanobacteriales (Methanobacterium, Methanothermobacter, Methanobrevibacter), Methanomicrobiales (Methanoculleus, Methanocorpusculum), Methanococcales and Methanocellales among them (Liu and Whitman 2008).
Their role in a digester is larger than their share of the methane. The bacteria that break down propionate and butyrate release hydrogen, and they can only keep doing so while hydrogen is removed as fast as it forms. Hydrogenotrophs are the organisms that remove it (Demirel and Scherer 2008). When they fall behind, volatile fatty acids accumulate: the familiar first sign of an upset has a hydrogen consumer behind it.
Methanoculleus deserves a name of its own. It is repeatedly found as the dominant hydrogen consumer of ammonia-rich digesters, where its tolerance of ammonia is part of why it persists as other methanogens decline (Westerholm, Moestedt, and Schnürer 2016; Wang, Fotidis, and Angelidaki 2015), and Methanoculleus bourgensis has been isolated from a biogas reactor (Maus et al. 2016). In the example report it carries the hydrogen route in both plants. In the BP3 reactor behind the depth tests in What one digestate sample can tell you, it was the only methanogen assembled at every depth from 2.5 to 12 Gb.
Only two groups of methanogens split acetate: Methanosarcina and the Methanotrichaceae: Methanothrix (formerly Methanosaeta) and Methanocrinis, which now holds the former Methanothrix harundinacea, a digester organism, alongside a newly described alkaliphilic species (Smith and Ingram-Smith 2007; Khomyakova et al. 2023). Because the route is carried by so few genera, which of them carries it in a given reactor says a good deal about that reactor.
Several relatives of Methanosarcina (Methanolobus, Methanococcoides, Methanomethylovorans) carry much of the same machinery but do not grow on acetate. A report credits the acetate route by genus, not by gene content alone, for that reason.
Methanothrix is the specialist. It uses nothing but acetate, grows slowly, and works at very low acetate concentrations that Methanosarcina cannot use (Jetten, Stams, and Zehnder 1992; Smith and Ingram-Smith 2007). It wins in a stable, well-fed reactor where acetate never accumulates. Methanosarcina is the generalist. It needs more acetate, grows faster, can use methyl compounds and, in most species, hydrogen, and tolerates high acetate, ammonia and salt far better, which has earned it the description of a methanogen for heavy-duty biomethanation (De Vrieze et al. 2012; Oren 2014). A survey of full-scale digesters found Methanothrix absent from reactors with high ammonia and volatile fatty acids, and Methanosarcina dominant in them (Karakashev, Batstone, and Angelidaki 2005).
That is the distinction the example report turns on. Plant A carries its acetate route on Methanothrix soehngenii at 3.7 % of mapped reads: abundant, efficient, and sensitive to ammonia. Plant B carries it on Methanosarcina flavescens at 0.49 %: scarce, but of a genus that tolerates what would stop plant A’s.
Under high ammonia the acetate splitters are inhibited, and acetate does not simply accumulate. Syntrophic bacteria oxidise it to hydrogen and CO2, and hydrogenotrophic methanogens turn those into methane (Schnürer and Nordberg 2008; Westerholm, Moestedt, and Schnürer 2016). The methane still comes from acetate, but through the hydrogen route, and the partnership depends on hydrogen being kept low, so it is slower and less forgiving than splitting acetate directly (Westerholm, Moestedt, and Schnürer 2016).
A community whose acetate route is thin and whose methanogens are mostly Methanoculleus is therefore consistent with a reactor running this way. DNA shows that the organisms and genes for it are present. It does not show that the pathway is carrying the flux, and a report says the first and not the second.
Methylotrophic methanogens make methane from methanol, methylamines and methylated sulfides (Söllinger and Urich 2019). In a digester these come from the feed: methanol from the demethylation of pectin, methylamines from the breakdown of choline, betaine and similar compounds (Söllinger and Urich 2019). The route is usually a minor share of the methane, but the organisms carrying it are more varied than for either of the other two.
Methylamine use depends on pyrrolysine, an amino acid encoded by a codon that normally means “stop”. Gene-calling software that stops there splits the gene in two, which is one reason the analysis calls its own genes.
Two kinds are worth telling apart. Some Methanosarcinales split methyl compounds on their own, among them Methanosarcina itself and the obligately methylotrophic Methanolobus, Methanococcoides and Methanomethylovorans (Oren 2014). Others reduce methyl groups with hydrogen and cannot make methane any other way: the Methanomassiliicoccales (Lang et al. 2015), Methanosphaera (Fricke et al. 2006), the Methanofastidiosa (Nobu et al. 2016) and the Verstraetearchaeota (Vanwonterghem et al. 2016). These consume hydrogen but cannot reduce CO2, so a report lists them under the methyl route; they still compete with the hydrogenotrophs for hydrogen. Methanogranum, which carries the methyl route in plant A of the example report, is one of the Methanomassiliicoccales.
| Genus | Route | What it tends to mean |
|---|---|---|
| Methanothrix | acetate | stable reactor, low acetate; sensitive to ammonia |
| Methanocrinis | acetate | the former Methanothrix harundinacea; a digester acetate specialist |
| Methanosarcina | acetate, methyl, most species hydrogen | tolerant of acetate, ammonia and salt; grows fast |
| Methanoculleus | hydrogen | common in ammonia-rich reactors; partner of acetate oxidisers |
| Methanobacterium, Methanothermobacter | hydrogen | hydrogen consumers; the latter thermophilic |
| Methanocorpusculum, Methanospirillum | hydrogen | hydrogen consumers |
| Methanomassiliicoccales (e.g. Methanogranum) | methyl, with hydrogen | methyl compounds in the feed |
A single name rarely decides anything. The useful reading is the combination: which genus carries each route, how much of it there is, and whether a route rests on one genome or several. A reactor whose acetate route rests on one ammonia-sensitive genome is in a different position from one where it rests on a tolerant genus, even if both are producing the same gas today.