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Review Article
Methylotrophic methanogens everywhere —
physiology and ecology of novel players in global
methane cycling
Andrea Söllinger* and Tim Urich
Institute of Microbiology, University of Greifswald, Felix-Hausdorff-Str. 8, 17487 Greifswald, Germany
Correspondence: Tim Urich (tim.urich@uni-greifswald.de)
Research on methanogenic Archaea has experienced a revival, with many novel lineages
of methanogens recently being found through cultivation and suggested via metage-
nomics approaches, respectively. Most of these lineages comprise Archaea (potentially)
capable of methanogenesis from methylated compounds, a pathway that had previously
received comparably little attention. In this review, we provide an overview of these new
lineages with a focus on the Methanomassiliicoccales. These lack the Wood–Ljungdahl
pathway and employ a hydrogen-dependent methylotrophic methanogenesis pathway
fundamentally different from traditional methylotrophic methanogens. Several archaeal
candidate lineages identified through metagenomics, such as the Ca.
Verstraetearchaeota and Ca. Methanofastidiosa, encode genes for a methylotrophic
methanogenesis pathway similar to the Methanomassiliicoccales. Thus, the latter are
emerging as a model system for physiological, biochemical and ecological studies of
hydrogen-dependent methylotrophic methanogens. Methanomassiliicoccales occur in a
large variety of anoxic habitats including wetlands and animal intestinal tracts, i.e. in the
major natural and anthropogenic sources of methane emissions, respectively. Especially
in ruminant animals, they likely are among the major methane producers. Taken together,
(hydrogen-dependent) methylotrophic methanogens are much more diverse and wide-
spread than previously thought. Considering the role of methane as potent greenhouse
gas, resolving the methanogenic nature of a broad range of putative novel methylotrophic
methanogens and assessing their role in methane emitting environments are pressing
issues for future research on methanogens.
Introduction
Methanogenesis, the biological formation of methane (CH
4
) by methanogens, is restricted to the
archaeal domain of life (e.g. [1–3]) although this view is currently challenged due to very recent
discoveries such as CH
4
production by Cyanobacteria[4] and aerobic CH
4
production by eukaryotes
[5,6]. Methanogens are an ecologically diverse group of microorganisms occurring in a great variety of
terrestrial and aquatic natural and anthropogenic anoxic environments, including wetlands, marine
and freshwater sediments, and gastro-intestinal tracts (GITs) of animals. Methanogens produce
CH
4
as an end-product of their anaerobic respiration [1]. Methanogenesis contributes to ∼70% of
present-day atmospheric CH
4
emissions [3]. Thus, methanogens are the major producers of the
potent greenhouse gas (GHG) CH
4
, which is responsible for ∼20% of the warming since pre-industrial
times [7].
Methanogenesis is a terminal process in anaerobic biomass degradation, common in habitats where
terminal electron acceptors, such as oxygen, nitrate, iron(III), and sulfate, are missing or rapidly
depleted [1,3]. The major substrates for methanogenesis are CO
2
, acetate, and methylated compounds.
*Current address: Department
of Arctic and Marine Biology,
UiT, The Arctic University of
Norway, Framstredet 39, 9019
Tromsø, Norway
Version of Record published:
10 December 2019
Received: 9 September 2019
Revised: 20 November 2019
Accepted: 21 November 2019
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Biochemical Society Transactions (2019)
https://doi.org/10.1042/BST20180565
Hence, three main methanogenesis pathways are distinguished: (i) CO
2
-reducing (hydrogenotrophic), (ii) aceti-
clastic, and (iii) methylotrophic methanogenesis (Figure 1). The last step in methanogenesis, the reduction of
methyl-coenzyme M to CH
4
performed by the methyl-coenzyme M reductase (Mcr) and the formation of the
coenzyme M–coenzyme B heterodisulfide (CoM-S-S-CoB), is conserved in all three pathways (Figure 1). The
subsequent reduction in the heterodisulfide by the heterodisulfide reductase (Hdr) to regenerate coenzyme M
(CoM) and coenzyme B (CoB) (Figure 1) is exergonic and coupled to the reduction in ferredoxin (Fd) and/or
energy conservation (see e.g. the reviews by Thauer et al. [2] and Lyu et al. [3] for more details). (i) The reduc-
tion of CO
2
to CH
4
is also called hydrogenotrophic methanogenesis, as hydrogen (H
2
) is the predominant elec-
tron donor (Figure 1). Alternatively, formate and more rarely, secondary alcohols, ethanol, and carbon
monoxide are also used as electron donors [1]. Hydrogenotrophic methanogenesis is the most common meth-
anogenesis pathway, occurring within six out of eight validly described methanogenic archaeal orders
Figure 1. Methanogenesis pathways.
Overview depicting the methanogenesis pathways found in Archaea, i.e. hydrogenotrophic, aceticlastic, methylotrophic, and
H
2
-dependent methylotrophic methanogenesis with focus on the H
2
-dependent methylotrophic methanogenesis of
Methanomassiliicoccales and the occurrence of methylotrophic methanogenesis specific enzymes in novel (putative)
methylotrophic lineages. Physiological, biochemical or genomic evidence for specific enzymes are indicated with colored
circles. Color code highlights selected parts of specific methanogenesis pathways as well as enzyme presence (evidence)
within selected archaeal lineages. For details and a comprehensive list of enzymes and compounds, see the main text and list
of abbreviations at the end of the main text. The presence of specific methanogenesis enzymes in Methermicoccus were
obtained from IMG annotation tables (https://img.jgi.doe.gov/; accessed the 21st of October, 2019). The presence of specific
enzymes in all other depicted methanogens were obtained from the references cited in the main text (in the respective
sub-chapters).
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Biochemical Society Transactions (2019)
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(Figure 2): Methanobacteriales,Methanococcales,Methanomicrobiales,Methanosarcinales,Methanopyrales, and
Methanocellales [1,2,8]. (ii) In aceticlastic methanogenesis (Figure 1), acetate (CH
3
-COO
−
) is split and the
carboxyl-group is oxidized to CO
2
while the methyl-group is reduced to CH
4
. Aceticlastic methanogenesis is
the least common methanogenesis pathway and only present within the order Methanosarcinales. However,
aceticlastic methanogenesis accounts for approximately two-thirds of global biogenic CH
4
production [1].
(iii) In methylotrophic methanogenesis, the methyl-groups of methylated compounds such as methanol,
methylamines, and methylated sulfides are transferred to substrate-specific corrinoid proteins and further
to CoM to finally be reduced to CH
4
(Figure 1). In most validly described methylotrophic methanogens
(mainly from the order Methanosarcinales), the required electrons for the reduction of the methyl-groups
are obtained via the oxidation of additional methyl-groups to CO
2
, with the exception of Methanomicrococcus
blatticola (Methanosarcinales) and the genus Methanosphaera (Methanobacteriales), which are methylotrophic
methanogens that employ external H
2
as an electron donor [1,2]. Methylotrophic methanogens are major
contributors to CH
4
production in marine sediments where hydrogenotrophic and aceticlastic methanogens
are outcompeted by sulfate-reducing bacteria [1,3]. Additionally, methylotrophic methanogens have also
been known from GITs, and extreme environments, e.g. hypersaline microbial mats and soda lake sediments
[1,9–11].
Figure 2. Methanogen diversity.
Schematic phylogenetic tree depicting major archaeal lineages. Tree topology is based on the extensive review on archaeal diversity, ecology, and
evolution by Spang et al. [75]. Methanogenic and putative methanogenic groups are highlighted in bold fonts. Colored dots and color code indicate
the methanogenesis pathway(s) carried out within a certain group. The asterisk indicates the poorly understood methanogenesis pathway of
Methermicoccus, which are capable of reducing methylated and methoxylated compounds. The question mark indicates the uncertainty regarding
the methane metabolism of Ca. Bathyarchaeota. ANME, anaerobic methane oxidizing Archaea; DPANN, proposed archaeal superphylum
comprising Diapherotrites, Parvarchaeota, Aenigmarchaeota, Nanohaloarchaeota, Nanoarchaeota, Woesearchaeota, Pacearchaeota, and
Micrarchaeota.
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H
2
-dependent methylotrophs —novel players in the global
CH
4
cycle
Until recently little attention was paid to methylotrophic methanogenesis as it was presumed to be less
common among methanogens than hydrogenotrophic methanogenesis and globally less important than aceti-
clastic methanogenesis. However, this perception has changed in the last years since the discovery of the
Methanomassiliicoccales [12–16]. In the following section, we first introduce the Methanomassiliicoccales,
describe their novel methanogenesis pathway, and chronologically present five other recently discovered
archaeal groups, all of which comprising putative methylotrophic methanogens.
Methanomassiliicoccales —methanogens without Wood–Ljungdahl pathway
Since the late 1990s, Thermoplasmata-related 16S rRNA gene sequences have been found in various environ-
ments ranging from the ocean [17,18] and rice fields [19] to the GITs of various animals and humans (e.g.
[20–23]). The co-occurrence of mcrA gene sequences (encoding the alpha subunit of Mcr) only distantly
related to characterized methanogens in several anaerobic environments suggested that some of these 16S
rRNA gene sequences might stem from methanogens. In 2012, Paul et al. [13] eventually described the group
as methanogens, based on a methanogenic enrichment culture and comparative 16S rRNA and mcrA gene ana-
lysis. In parallel, the first validly described and still sole isolate of this group, Methanomassiliicoccus luminyensis
was reported [12]. Subsequently, Methanomassiliicoccales was given as the officially accepted name to the
seventh order of methanogenic Archaea [15,16].
Like their close relatives, the Thermoplasmatales,Methanomassiliicoccales lack a rigid cell wall; instead, they
are surrounded by two membranes [12,24,25]. Remarkably, M. luminyensis comprises a unique membrane
lipid inventory, e.g. glycerol sesterpanyl-phytanyl diether core lipids (mainly found in halophilic Archaea) and
compounds bearing either heptose or methoxylated glycosidic head groups (for the first time reported for
Archaea). Most strikingly, M. luminyensis contains fundamentally novel tetraether lipids, in which one glycerol
(C3) backbone is replaced by either butane triol (C4) or pentane triol (C5) [26].
Based on physiological, genomic, and metatranscriptomic data Methanomassiliicoccales employ a methylo-
trophic methanogenesis pathway reducing methanol, methylamines, and presumably also methylated sulfides to
CH
4
(Figure 1)[24,25,27,28]. Genomics furthermore revealed that Methanomassiliicoccales lack the methyl-
branch (MB) of the Wood–Ljungdahl (WL) pathway (Figure 1). Like M. blatticola and Methanosphaera
species, Methanomassiliicoccales employ external H
2
as an electron donor (Figure 1), extending thus the spec-
trum of organisms with H
2
-dependent methylotrophic methanogenesis (Figure 2).
H
2
-dependent methylotrophic methanogenesis —Methanomassiliicoccales as
an emerging model group
The Methanomassiliicoccales have in recent years developed into a model for the H
2
-dependent methylotrophic
methanogens. Nevertheless, and despite many efforts, only one member of the Methanomassiliicoccales could
be obtained as pure culture and validly described so far, M. luminyensis [12]. However, several enrichments
and draft genomes have been published in the last years [13–15,25,28–32]. Together with cell-biological and
physiological studies on M. luminyensis [26,33,34] as well as environmental metatranscriptomics studies
[27,35], these studies provided first remarkably insights into the cell biology, physiology, and ecology of
Methanomassiliicoccales, making them a treasure trove for the discovery of fundamentally new biology.
Besides M. luminyensis, several Methanomassiliicoccales genomes obtained from enrichment metagenomes
have been published in the last few years (Figure 3). Enrichments, all of which grown on methanol or trimethy-
lamine (TMA) and H
2
as methanogenesis substrates, were obtained from termite and millipede guts [13,25],
human gut [14,29], an anaerobic digester [15], and bovine [28,30] and ovine rumen [31,32]. Comparative gen-
omics revealed that all Methanomassiliicoccales genomes lack the entire MB of the WL pathway (e.g.
[24,25,28]). Thus, Methanomassiliicoccales are not able to oxidize methyl-groups to CO
2
and are therefore
dependent on an external electron donor, namely H
2
(Figure 1). Consequently, Methanomassiliicoccales differed
fundamentally from all previously described methanogens. The MB of the WL pathway, together with the Mcr-
and Mtr-complex (Mtr, methyl-tetrahydromethanopterin:CoM methyltransferase complex), was thought to be
a common trait encoded by all methanogenic Archaea independent of their type of methanogenesis (recently
reviewed by Borrel et al. [36]). The carboxyl-branch of the WL pathway, leading to biomass formation out of
CO
2
and –CH
3
via acetyl-CoA, seems to be partly present in Methanomassiliicoccaceae. However, because of
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Biochemical Society Transactions (2019)
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structural differences, missing, fused and truncated subunits, and several insertions within subunits, it was pro-
posed that these genes may no longer encode a functional enzyme or if functional, it may serve to generate C1
compounds from acetyl-CoA [25,36]. Acetate appears to be the carbon source of Methanomassiliicoccales, and
all genomes encode an ADP-forming acetyl-coenzyme A synthetase and a pyruvate:Fd oxidoreductase (e.g.
[25,28]).
Furthermore, comparative genomics revealed that besides methanol and TMA, mono- and dimethylamine
(MMA and DMA), as well as methylated sulfides possibly, can be utilized as an electron acceptor by
Figure 3. Phylogeny, Ecology, and Physiology of Methanomassiliicoccales.
Overview summarizing all published Methanomassiliicoccales genomes and their placement within the two broad
Methanomassiliicoccales clades. Due to the limited number of genomes representing the environmental clade and the wetland
cluster representative sequences were selected from the SILVA database to equalize the numbers of sequences for each
group. The Maximum likelihood tree was inferred using IQ tree and the GTR + F + G4 + I substitution model [76] based on an
alignment of 948 16S rDNA positions (clustalW; bioedit; [77]). Node labels represent ultrafast bootstrap values (1000 replicates)
[78]. Information on the genomes, metabolic potential and capabilities, and (isolation) source were obtained from the literature,
see accession numbers and references. The dotted line indicates the wetland cluster. The tree is considered to give a
phylogenetic overview, not claiming to reflect the relationship of Methanomassiliicoccales within the broad groups.
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Methanomassiliicoccales (Figures 1 and 3). Growth experiments on M. luminyensis and Ca. Methanoplasma ter-
mitum largely confirmed these comparative genomic analyses. While M. luminyensis was able to grow on
methanol, MMA, DMA, and TMA [25,34], Ca. M. termitum only showed growth on methanol and MMA
[25]. The growth of Methanomassiliicoccales on methylated sulfides, such as dimethylsulfide (DMS), has not
yet been shown experimentally. However, proteome analysis of M. luminyensis cells grown on methanol and
H
2
showed the presence of MtsAB, methyltransferases specific for methylated sulfides (Söllinger, Schäfer and
Urich, unpublished). Nevertheless, they were much lower in abundance as compared with the methanol-specific
methyltransferases.
Like the previously described Methanosphaera spp. and M. blatticola,Methanomassiliicoccales are dependent
on H
2
as an electron donor. However, it is suggested that their energy conservation is different (Figure 1).
According to genomic and biochemical studies (e.g. [24,25,28,37]) the regeneration of CoM and CoB in
Methanomassiliicoccales seems to be coupled to a membrane-bound energy-converting Fd:heterodisulfide oxi-
doreductase complex (Fpo-like complex + HdrD) and a soluble methyl viologen-dependent hydrogenase/Hdr
complex (MvhADG/HdrABC) (Figure 1; for more details see Lang et al.[25]). In contrast Methanosphaera spp.
employ a soluble methyl viologen-dependent hydrogenase/Hdr complex (MvhADG/HdrABC) (e.g. [2]), while
M. blatticola appears to employ a membrane-bound Hdr system (i.e. HdrDE), possibly coupled to a
methanophenazine-reducing hydrogenase (Vho) typical for Methanosarcinales[38](Figure 1). No methanophe-
nazines or respiratory quinones were detected among the membrane lipids of M. luminyensis [26], offering
further biochemical evidence for a methanogenesis pathway different from the cytochrome-containing methylo-
trophic Methanosarcinales (Figure 1). Additionally, proteome analysis of M. luminyensis revealed the presence
of HdrD and several Fpo subunits, besides HdrABC and Mvh subunits (Söllinger, Schäfer and Urich, unpub-
lished). High expression levels of the genes encoding HdrABC and HdrD were also shown in M. luminyensis
via quantitative RT-PCR [33].
A genomic analysis of a Methanomassiliicoccales draft genome obtained from an oil mining tailing pond
metagenome, referred to as MALP (MAssiliicoccales Lake Pavin; Figure 3), was recently reported [39]. The
MALP genome lacks methylamine- and methylsulfide-specific methyltransferase genes but contains genes
which could possibly allow for aceticlastic methanogenesis, i.e. genes encoding acetyl-CoA synthetase ( forma-
tion of acetyl-CoA from acetate), acetyl-CoA synthase/CO dehydrogenase (disproportionation of acetyl-CoA
into methyl-tetrahydromethanopterin and CO), Fd dependent oxidation of CO to CO
2
, and the catalytic subu-
nits mtrAH of the methyl-tetrahydromethanopterin:CoM methyltransferase (Mtr). However, the authors
pointed out that this pathway is highly uncertain, since none of the membrane associated methyl-
tetrahydromethanopterin:CoM methyltransferase subunits (mtrB-G) are present, mtrA contains and unusual
C-terminal extension, mtrH is divergent from the mtrH of CO
2
-reducing methanogens, and tetrahydrometha-
nopterin is likely absent from MALP [39]. Obtaining further Methanomassiliicoccales isolates would be of great
value to gain deeper insights into their physiological versatility.
Methylotrophic methanogens everywhere
Metagenomics has tremendously added insights into the coding potential of microbial dark matter through
metagenome-assembled genomes (MAGs) of many yet uncultured lineages. Although physiological and/or bio-
chemical evidence for methylotrophic methanogenesis is missing, the studies have identified several novel
lineages of putative methylotrophic methanogens.
Candidatus Bathyarchaeota —non-euryarchaeal potential methane
metabolizers
In 2015, Evans et al. [40] reported two draft genomes (BA1 and BA2) of Candidatus (Ca.) Bathyarchaeota, a
yet uncultured archaeal candidate phylum formerly known as Miscellaneous Crenarchaeotal Group (MCG).
The MAGs were obtained from the biomass of coal-bed CH
4
wells. Surprisingly, comparative genome analysis
revealed the presence of genes encoding the Mcr-complex as well as genes for methylotrophic methanogenesis
from methanol, methylamines, and methylated sulfides similar to the Methanomassiliiicoccales [40], showing
for the first time genes encoding the methanogenesis pathway outside of the Euryarchaeota (Figures 1 and 2).
Evans et al. [40] hypothesized that at least BA1 might represent a methylotrophic methanogen capable of using
a range of methylated compounds. Ca. Bathyarchaeota are found in a broad range of anoxic environments
including marine sediments, soils, freshwater environments, and hot springs [41,42]. Recently the metabolic
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diversity within the Ca. Bathyarchaeota has been reviewed [42–44], and it is suggested that the organisms
might rather be anaerobic methane or short-chain hydrocarbon oxidizers that employ a reverse methanogenesis
pathway.
Ca. Methanofastidiosa —putative fastidious methylotrophic methanogens
Similar to the Methanomassiliicoccales, the methanogenic nature of another uncultured archaeal lineage
(WSA2/Arc I, potentially a class level clade within the Euryarchaeota) was suspected for years, mainly due to
its presence in methanogenic environments [45–47]. In 2016, Nobu et al. [48] reported the first WSA2 draft
genomes, obtained from the anaerobic digester and methanogenic bioreactor metagenomes. Comparative gen-
omics suggested that Ca. Methanofastidiosa are H
2
-dependent methylotrophic methanogens restricted to
methylated thiol compound reduction [48]. Thus, the name Ca. Methanofastidiosa was proposed to highlight
their unique limited metabolic capacity. Like Methanomassiliicoccales,Ca. Methanofastidiosa lack the WL
pathway and the Mtr-complex (methyltetrahydromethanopterin:CoM methyltransferase complex; Figure 1),
challenging the generality of the classical association of methanogenesis and the WL pathway [36]
Ca. Verstraetearchaeota —a proposed phylum comprising methylotrophic
methanogens
With the discovery of putative methanogens within another Candidatus phylum, Ca. Verstraetearchaeota in
2016 the dogma of methanogenesis originating within the phylum Euryarchaeota and restricted to
Euryarchaeota was challenged again [49]. Initially, one candidate order was described, Ca. Methanomethyliales
and it was proposed that the methanogenesis pathway of its members might be similar to
Methanomassiliicoccales and Ca. Methanofastidiosa [49], which would expand methanogenic diversity and the
distribution of H
2
-dependent methylotrophic methanogenesis (Figures 1 and 2). Members of the Ca.
Methanomethyliales encode genes to reduce methanol, methylated sulfides, and methylamines to CH
4
[49].
Very recently two novel orders within the Verstraetearchaeota were proposed, Ca. Methanomediales and Ca.
Methanohydrogenales [50]. While Ca. Methanomediales represent presumably H
2
-dependent methylotrophic
methanogens, a draft genome of the latter encodes genes for all key enzymes of hydrogenotrophic methanogen-
esis, expanding the putative methanogenesis diversity within the Ca. Verstraetearchaeota.
Methermicoccus —coal-eating methoxydotrophs
Methermicoccus shengliensis, a thermophilic methylotroph is the first and still sole isolated representative of the
Methermicoccaceae family within the order Methanosarcinales[51]. Although formally described already in
2007, it received wider attention in 2016 when it was shown that M. shengliensis is able to produce CH
4
from
over 30 types of methoxylated aromatic compounds and coal [52], expanding the substrate range of methylo-
trophic methanogens. Therefore, M. shengliensis not only represents the methanogen with the highest metabolic
flexibility found to date (Figure 1), but also represents the first organism that is able to perform the complete
anaerobic demethylation of methoxylated compounds, which was thought to be exclusively performed by syn-
trophic consortia of acetogens and methanogens [53]. This novel methoxydotrophic methanogenesis might
play an important role in deep surface environments and biogenic gas formation including coal-bed methane
[52].
Methanonatronarchaeia —extremely halophilic methylotrophs
Methanogenic enrichments from hypersaline lakes led in 2017 to the discovery of a very remarkable class
level-group of methylotrophic methanogens that are extremely halo(natrono)philic and related to the class
Haloarchaea (known as Halobacteria)[54]. So far, Sorokin et al. [55] reported eleven isolates from soda lakes
and three enrichments from salt lakes, all of which grew optimally at 50°C, 4 M Na
+
at alkaline and neutral
pH, respectively. It was shown that the isolates reduce methylated compounds such as methanol, methylamines,
and methylated sulfides to CH
4
, with formate or hydrogen as an electron donor (Figure 1). Unlike other salt-
tolerant and halophilic methanogens, all of which are members of the Methanosarcinales, the
Methanonatronarchaeia use the ‘salt-in’osmoprotection mechanism, similar to their relatives, the Haloarchaea.
Very recently, also within members of the phylum Korarchaeota genes encoding a methanogenesis pathway
similar to the one in Methanomassiliicoccales were discovered [44,56]. Notably, all of the above-mentioned
novel methanogens appear to reduce methylated compounds to CH
4
. Even more strikingly, nearly all of them
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employ hydrogen as an external electron donor and the methylated compounds only act as electron acceptors.
Thus, methylotrophic methanogenesis, especially H
2
-dependent, is much more common among methanogens
than previously assumed. Furthermore, these methylotrophs are found in a large diversity of natural and
anthropogenic anoxic environments including wetlands, ruminant animals, marine and freshwater sediments,
anaerobic digesters, hypersaline, thermophilic, and deep subsurface environments. However, especially physio-
logical insights are hampered due to the lack of isolated representatives, with the exception of the
Methanomassiliicoccales and the Methanonatronarchaeia [12,55].
Ecology
The ecological importance of most of the above introduced (putative) methylotrophs has not yet been
addressed. First, the methanogenic nature of these groups needs to be proven by physiological studies and culti-
vation. However, attempts have been made to assess the ecological role and importance of
Methanomassiliicoccales.
Methanomassiliicoccales are found in a great variety of natural and anthropogenic anoxic environments
including wetlands and GITs of ruminant animals, the major natural and a major anthropogenic CH
4
emission
sources, respectively. Based on differential but non-exclusively habitat preferences Methanomassiliicoccales were
divided into two broad clades [28], a GIT clade containing most of the currently available
Methanomassiliicoccales enrichment cultures and an environmental clade, including M. luminyensis and a
wetland-specific cluster (Figure 3). Although M. luminyensis,Ca. M. intestinalis, and RumEn M1 were isolated
from human feces and bovine rumen fluid, respectively, they are representatives of the environmental clade,
revealing a substantial enrichment bias [28]. A dominance of GIT clade members in human feces and bovine
rumen fluid has been reported [23,35,57]. In general, GIT clade members appear to have smaller genome sizes
compared with environmental clade Methanomassiliicoccales (Figure 3). Assumingly, the dominant
Methanomassiliicoccales in anoxic environments other than animal GITs face higher variations in environmen-
tal parameters such as moisture, salinity, oxygen exposure, and substrate/nutrient availability than GIT
Methanomassiliicoccales, which is reflected by a higher number of genes encoding enzymes for osmoprotection,
oxidative stress response, and the potential for diazotrophy within environmental clade genomes [24,28].
Ruminant animals
Methanomassiliicoccales constitute a major proportion of the methanogens in the rumen [58–60]. In metatran-
scriptomic studies of the bovine rumen microbiome, it was shown that they were highly active and abundant
[27], especially after feed uptake [35]. Methanomassiliicoccales were also identified as transcriptionally active in
the ovine rumen [61,62]. Due to their capability of utilizing methylamines as methanogenesis substrates,
Methanomassiliicoccales occupy a previously unnoticed ecological niche in the rumen [27]. Furthermore,
ammonium is produced as an end-product of methylamine reduction and likely serves as a nitrogen source for
many other rumen microorganisms [27]. In contrast, they compete with other methanogens for the electron
donor H
2
and they compete for methanol with Methanosphaera species. Recently, we showed that
Methanomassiliicoccales transcripts for methylamine-specific methyltransferases increased while methanol-
specific methyltransferases decreased immediately after feed intake [35]. Bevor feeding, six times more
methylamine-specific methyltransferase transcripts were present, as compared with methanol-specific methyl-
transferase transcripts. This ratio increased to 30-fold immediately after feeding. In contrast, Methanosphaera
spp. methanol-specific methyltransferase transcripts increased after feeding [35]. Thus, Methanomassiliicoccales
may avoid the competition with Methanosphaera spp. by utilizing methylamines, when available.
In general, hydrogenotrophic methanogens, mainly Methanobrevibacter sp. (Methanobacteriales), are consid-
ered as dominant CH
4
producers in ruminants [1]. Furthermore, CO
2
is a much more abundant methanogen-
esis substrate than methylamines and methanol. Thus, it is somewhat surprising that Methanomassiliicoccales
are so abundant and active (see below, perspectives). Intensified agriculture is currently considered the main
cause of increasing atmospheric CH
4
concentrations [63]. As the demand for milk and meat is expected to
strongly increase by 2050 [64], CH
4
emissions from agriculture will likely also continuously increase. Taking
this into account, rumen Methanomassiliicoccales appear as potent targets for CH
4
mitigation strategies [27,35].
In fact, Poulsen et al. [27] identified rapeseed oil as an effective compound to inhibit CH
4
formation by
Methanomassiliicoccales. However, the mode of action is not yet understood.
© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society8
Biochemical Society Transactions (2019)
https://doi.org/10.1042/BST20180565
Wetlands
In wetlands, the major source of natural CH
4
emission, Methanomassiliicoccales of the wetland cluster are also
widely distributed [28]. However, not much is known about their abundance and activity. Analysis of metatran-
scriptomes from two arctic wetlands on Svalbard, Norway [65] revealed low relative abundance of
Methanomassiliicoccales 16S rRNA transcripts; up to 4.7% of all methanogens [28]. However, a recent study on
methanogenic communities in Tibetan high elevation wetland soils identified Methanomassiliicoccales as the
second most abundant and diverse methanogenic group, with relative abundances of up to 60% [66].
Therefore, the authors concluded an important, but potentially overlooked, role of Methanomassiliicoccales in
CH
4
production in wetlands. Additionally, we detected Methanomassiliicoccales in three different mire types in
Eastern Germany, where they constituted up to 50% of all methanogenic Archaea in a high-methane emitting
percolation mire (Weil, Söllinger and Urich, unpublished), indicating a possibly important role of
Methanomassiliicoccales in some temperate peatlands.
The recently published near-complete MALP genome [39], closely related to wetland cluster
Methanomassiliicoccales, seems to be very reduced (Figure 3). The MALP genome encodes the genes necessary
for the reduction in methanol to CH
4
but none of the genes necessary for the reduction in other methylated
compounds. However, Speth and Orphan [39] raise the possibility of aceticlastic methanogenesis from the com-
parative genome analysis of MALP. Obtaining further genomes and ideally isolates is inevitable to gain insights
into the physiology and the substrate spectrum of this much-understudied group within the
Methanomassiliicoccales [1,3].
Even less is known about the abundance and activity of Methanomassiliicoccales in other terrestrial and
aquatic anoxic environments, although the presence of mcrA sequences, later identified as
Methanomassiliicoccales sequences (e.g. in [13]), was shown in a great variety of anoxic habitats including rice
fields ([19]; rice cluster III), landfill soil [67], lakes [68,69], and riparian soil [70]. They have also been reported
as members of the methanogenic communities in hypersaline mats [71,72]. In conclusion, the order
Methanomassiliicoccales appear to be a rather heterogeneous and widespread group. Further isolates, especially
from the wetland cluster and the GIT clade, but also from other environments, are much-needed to better
understand the (eco-)physiological basis of their ecological success.
Perspectives
The numerous discoveries described above are currently putting a focus on (H
2
-dependent)
methylotrophic methanogens, showing that their phylogenetic diversity is much larger than previ-
ously assumed. Their global relevance for the carbon/CH
4
cycle has to date not been sufficiently
addressed. However, the emerging model group Methanomassiliicoccales can be highly abun-
dant and transcriptionally active especially in ruminant animals [27,35,61,62], indicating that they
might be major contributors to global atmospheric CH
4
emissions. Therefore,
Methanomassiliicoccales appear as potential targets for future CH
4
mitigation strategies in agri-
culture [35]. Furthermore, the human-associated Methanomassiliicoccales are considered to play
a role in human health and disease, as their capability to reduce TMA to CH
4
could be used to
reduce TMA concentrations within the human GIT, which are associated with cardiovascular
disease and the metabolic disorder trimethylaminuria [73,74].
Future directions should include research on the sources of methyl substrates, especially
important in the rumen system. Who are the interactions partners? What sustains the high abun-
dance of Methanomassiliicoccales? What fermentative pathways have potentially been over-
looked? In fact, research on rumen methanogens did not miss anything before their discovery.
Thus, an explicit link to the process of methylotrophic methanogenesis in situ would be desirable.
Alternatively, the metabolic versatility of Methanomassiliicoccales has not been fully elucidated
and alternative metabolisms besides methylotrophic methanogenesis might be carried out.
Furthermore, their relationship to H
2
should be a research focus, since they are in competition
for H
2
with other microorganisms, including e.g. hydrogenotrophic methanogens, such as
Methanobrevibacter sp. in the animal GITs. One reason for the high abundance in the rumen
might be the comparably high H
2
partial pressure. However, hydrogenotrophic methanogenesis
is thermodynamically more favorable than e.g. H
2
-dependent methylotrophic methanogenesis
© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society 9
Biochemical Society Transactions (2019)
https://doi.org/10.1042/BST20180565
from methanol under standard conditions (−135 kJ mol
−1
vs. −113 kJ mol
−1
CH
4
, respectively
[1]). Nevertheless, the affinity towards H
2
might differ between the methanogens. The influence of
H
2
partial pressure on Methanomassiliicoccales has not been addressed yet. Lang et al. [25] sug-
gested that the energy metabolism of Methanomassiliicoccales may serve to increase their affinity
for H
2
, however, experimental verification is lacking.
Clearly, more representatives need to be brought into the culture for physiological and bio-
chemical studies. Among the Methanomassiliicoccales, the GIT clade and the wetland cluster are
of particular interest. In a wider perspective, it would be highly desirable to have representative
isolates or enrichment cultures of the other new putative methylotrophic methanogenic Archaea.
In conclusion, the here presented example illustrates impressively the power of enrichment cul-
tivation in the meta-omics era, where one new isolate with still rather poorly characterized physi-
ology paves the way for an avalanche of metagenomic discoveries. Undoubtedly, more
biochemistry and physiology studies will reveal more exciting insights into the physiology and
environmental adaptations of hydrogen-dependent methylotrophic methanogens.
Abbreviations
ADP, adenosine diphosphate; ATP, adenosine triphosphate; CH
3
-CoM, methyl-coenzyme M; CH
3
-COO−,
acetate; CH
3
-H
4
MPT, methyl-tetrahydromethanopterin; CH
4
, methane; CO
2
, carbon dioxide; CoB-SH, coenzyme
B; CoM-SH, coenzyme M; CoM-S-S-CoB, coenzyme M–coenzyme B heterodisulfide; DMA, dimethylamine;
DMS, dimethylsulfide; Ehb, energy-converting hydrogenase; Fd, ferredoxin; Fd
red
2−
, reduced Fd; Fpo,
membrane-bound F
420
-methanophenazine oxidoreductase complex; Fpo-like/HdrD, membrane-bound
energy-converting ferredoxin:heterodisulfide oxidoreductase complex; GHG, greenhouse gas; GIT,
gastro-intestinal tract; H
+
, proton; H
2
, hydrogen; Hdr, heterodisulfide reductase; HdrDE, membrane-bound
heterodisulfide reductase; MAGs, metagenome-assembled genomes; MALP, MAssiliicoccales Lake Pavin; MB,
methyl-branch; Mcr, methyl-coenzyme M reductase; mcrA gene, gene encoding the alpha subunit of Mcr;
MePh, methanophenazine; MMA, monomethylamine; MtaAB, methanol-specific methyltransferase; MtbA,
methylamine-specific methyltransferase; MtbB, DMA-specific methyltransferase; MtbC, DMA-specific corrinoid
protein; MtmB, MMA-specific methyltransferase; MtmC, MMA-specific corrinoid protein; Mtr,
methyl-tetrahydromethanopterin:CoM methyltransferase; MtsAB,, methyltransferases specific for methylated
sulfides; MttB, TMA-specific methyltransferase; MttC, TMA-specific corrinoid protein; Mvh(ADG)/Hdr(ABC),
soluble methyl viologen-dependent hydrogenase/heterodisulfide reductase complex; Na
+
, sodium ion; NH
4
,
ammonium; RT-PCR, reverse transcription polymerase chain reaction; TMA, trimethylamine; Vho,
methanophenazine-reducing hydrogenase; Wood-Ljungdahl pathway; WL pathway, Wood–Ljungdahl pathway.
Author Contributions
T.U. designed research. A.S. and T.U. wrote the manuscript. A.S. created the figures.
Funding
The authors acknowledge financial support by the European Social Fund (ESF) and the Ministry of Education,
Science and Culture of Mecklenburg-Western Pomerania within the scope of the projects WETSCAPES (ESF/
14-BM-A55-0032/16) and KOINFEKT (ESF/14-BM-A55-0013/16). A.S. was financially supported by a scholarship
from the University of Vienna for doctoral candidates (uni:docs) and a scholarship by the OeAD (Austrian Agency
for International Mobility and Cooperation in Education, Science and Research) for doctoral candidates
(Marietta-Blau-Fellowship).
Competing Interests
The authors declare that there are no competing interests associated with the manuscript.
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