Discovery of extremely halophilic, methyl-reducing euryarchaea provides insights into the evolutionary origin of methanogenesis

Over the last few years, the methyl-reducing pathway of methanogenesis, from an inconsequential curiosity, became one of the major themes in the methanogenesis research field

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This hybrid pathway was found in several deep lineages of methanogens, even outside the phylum Euryarchaeota, to which all the well-characterized methanogens belong. So far, however, only a single organism executing this pathway has been isolated and characterized in pure culture. We identified a deep lineage of methyl-reducers in hypersaline lakes that appear to represent a new class of the Euryarchaeota that is mostly closely related to the haloarchaea. These methanogens are extreme halo(alkali)philes and moderate thermophiles. This discovery changes the established view on methanogenesis in hypersaline habitats as the exclusive domain of classical methylotrophs. 

The fundamental knowledge on the diversity, physiology and biochemistry of methanogenic Euryarchaeota is quite extensive, with three major pathways (hydrogenotrophic, methylotrophic and acetoclastic) well established in 6 orders of methanogenic eurychaea represented in culture. However, for a long time,  there has been no major progress in this field. And now, look at what happened in the last 5 years - a real explosion!

It started with a paper describing a methanogen from the animal guts with a strange name Methanomassiliicoccus (Dridi et al., 2102), which I missed completely. Only after several follow-up papers on phylogeny and genomics of several additional members of this new, 7th order of methanogens appeared in rapid succession, they started to attract more and more attention, and became an exciting new line of methanogenesis research (Borrel et al., 2013; 2014; Lang et al., 2015). Subsequent  metagenomic analysis led to the discovery of 3 more deep lineages, two of them even outside of the Euryarchaeota, for which genomic reconstruction indicated the same type of metabolism as identified in Methanomassiliicoccales (Evans et al., 2015; Nobu et al., 2015; Vanwonterghem et al., 2016). In this 4-th pathway, called methyl-reduction, only the last of the classical 7 steps is present (Mcr), whereas the other 6 enzymes converting CO2 to methyl group in hydrogenotrophs, or vice versa in methylotrophs, are lacking. It has already been discovered a long time ago in two methanogens but was considered an inconsequential curiosity. Now it has become clear that it is at least as prominent as the other 3 classical pathways. 

For the last 5 years, I was investigating extremophilic methanogenic archaea in soda lakes - a unique type of salt lakes with a very high soluble carbonate alkalinity maintaining constant high pH around 10. 
(a photograph of a hypersaline soda lake in s-w Siberia). While performing activity tests with sediments and Mcr profiling in 2012, we identified a deep Mcr lineage, which did respond to the addition of typical methanogenic substrates. Next year I started to hunt that lineage by manipulating the incubation conditions and, finally, got it! 

It already became clear, at that stage, that something very unusual was "struggling on my fishing hook", because it required a combination of conditions in which the soda lake methanogens cultivated before would not grow: saturated soda brines with pH 10, high temperature and two substrates, a C1 methylated compound and either formate or H2, hinting at the methyl-reducing pathway. The next step was to try to cultivate these organisms. It worked for 3 successive 1:100 transfers - and then stopped. 

The activity disappearance corresponded to complete dilution of the initial sediment material from the enrichments. Addition of a sterilized anaerobic sediment slurry from the same lakes to an inactive culture resulted in explosive formation of methane. Further attempts to replace the sediments with less exotic compounds finally showed that the culture can be grown with additions of CoM, small amount of yeast extract and colloidal FeS, on top of MeOH/formate as the main substrates. Light microscopy showed domination of tiny cocci barely visible at the highest magnification, which suggested the possibility of purification by filtration. And it worked! Using this approach, 11 pure cultures were isolated from various soda lakes in 2 years. Both in the case of FeS only and the sediment-containing cultures, the growth always started in the solid phase where the methanogens formed a biofilm. Particularly spectacular mineral-associated methanogenesis was observed in the presence of FeS. (video file) But the true nature of this obligate dependence on FeS or sediments in these methanogens is still obscure. 

So, that is the actual story, the rest is more or less in the paper. Shortly, the soda lake group of isolates was joined by highly enriched cultures of its neutrophic counterpart from hypersaline salt lakes and, together, they formed a novel deep phylogenetic lineage of extremely halophilic and moderately termophilic  methyl-reducing methanogens related to haloarchaea. The genomic and evolutionary reconstructions performed by my colleagues at the NCBI showed two principal differences from the known methyl-reducers:  (1) the presence of a respiratory chain similar to that in the cytochrome-encoding members of the Methanosarcinales and (2) a presence of 4 of the 6 genes encoding the reversible CO2 reduction to methane pathway. These suggest a distinct evolutionary history of the methyl-reducing pathway. 

Many questions remain to be answered regarding this type of methanogens. The most important one, in my opinion, is how they compete with the classical methylotrophs for the C1 compounds and with the hydrogenotrophs (not only methanogens but also SRP and homoacetogens) for formate/H2? And another one, of course, is the evolutionary scenario for the different types of methanogenesis that remains unclear.  

The paper in Nature Microbiology is here:


  • Borrel G. et al. (2013) Phylogenomic data support a seventh order of methylotrophic methanogens and provide insights into the evolution of methanogenesis. Genome Biol Evol 5: 1769-1780.
  • Borrel G. et al. (2014) Comparative genomics highlights the unique biology of Methanomassiliicoccales, a Thermoplasmatales-related seventh order of methanogenic archaea that encodes pyrrolysine. BMC Genom 15: 679.
  • Lang K. et al. (2015) New mode of energy metabolism in the seventh order of methanogens as revealed by comparative genome analysis of "Candidatus methanoplasma termitum". Appl Environ Microbiol 81: 1338-1352
  • Evans PN et al. (2015) Methane metabolism in the archaeal phylum Bathyarchaeota revealed by genome-centric metagenomics. Science 350: 434-438.
  • Nobu MK et al. (2016) Chasing the elusive Euryarchaeota class WSA2: genomes reveal a uniquely fastidious methylreducing methanogen. ISME J 10: 2478-24-87. 
  • Vanwonterghem I et al. (2016) Methylotrophic methanogenesis discovered in the archaeal phylum Verstraetearchaeota. Nat Microbiol 1: article 16170.

Dimitry Y Sorokin

senior scientist, TU Delft