Methanogen

Methanogens are anaerobic archaea that produce methane as a byproduct of their energy metabolism, i.e., catabolism. Methane production, or methanogenesis, is the only biochemical pathway for ATP generation in methanogens. All known methanogens belong exclusively to the domain Archaea, although some bacteria, plants, and animal cells are also known to produce methane. However, the biochemical pathway for methane production in these organisms differs from that in methanogens and does not contribute to ATP formation. Methanogens belong to various phyla within the domain Archaea. Previous studies placed all known methanogens into the superphylum Euryarchaeota. However, recent phylogenomic data have led to their reclassification into several different phyla. Methanogens are common in various anoxic environments, such as marine and freshwater sediments, wetlands, the digestive tracts of animals, wastewater treatment plants, rice paddy soil, and landfills. While some methanogens are extremophiles, such as Methanopyrus kandleri, which grows between 84 and 110 °C, or Methanonatronarchaeum thermophilum, which grows at a pH range of 8.2 to 10.2 and a concentration of 3 to 4.8 M, most of the isolates are mesophilic and grow around neutral pH.

Physical description

Methanogens are usually cocci or rods in shape, but long filaments and curved forms also occur. There are over 150 described species of methanogens, which do not form a monophyletic group in the phylum Euryarchaeota. They are exclusively anaerobic organisms that cannot function under aerobic conditions due to the extreme oxygen sensitivity of methanogenesis enzymes and FeS clusters involved in ATP production. However, the degree of oxygen sensitivity varies, as methanogenesis has often been detected in temporarily oxygenated environments such as rice paddy soil, and various molecular mechanisms potentially involved in oxygen and reactive oxygen species detoxification have been proposed. For instance, a recently identified species Candidatus Methanothrix paradoxum common in wetlands and soil can function in anoxic microsites within aerobic environments but it is sensitive to the presence of oxygen even at trace level and cannot usually sustain oxygen stress for a prolonged time. However, Methanosarcina barkeri from a sister family Methanosarcinaceae is exceptional in possessing a superoxide dismutase enzyme, and may survive longer than the others in the presence of O₂.
As is the case for other archaea, methanogens lack peptidoglycan, a polymer that is found in the cell walls of bacteria. Instead, some methanogens have a cell wall formed by pseudopeptidoglycan. However, most of methanogens have a paracrystalline protein array that fits together like a jigsaw puzzle. In some lineages there are less common types of cell envelope such as the proteinaceous sheath of Methanospirillum or the methanochondroitin of Methanosarcina aggregated cells.

Ecology

In anaerobic environments, methanogens play a vital ecological role, removing excess hydrogen and fermentation products that have been produced by other forms of anaerobic respiration. Methanogens typically thrive in environments in which all electron acceptors other than CO₂ have been depleted. Such environments include wetlands and rice paddy soil, the digestive tracts of various animals, wastewater treatment plants and landfills, deep-water oceanic sediments, and hydrothermal vents. Most of these environments are not categorized as extreme, and thus the methanogens inhabiting them are also not considered extremophiles. However, many well-studied methanogens are thermophiles such as Methanopyrus kandleri, Methanothermobacter marburgensis, Methanocaldococcus jannaschii. On the other hand, gut methanogens such as Methanobrevibacter smithii common in humans or Methanobrevibacter ruminantium omnipresent in ruminants are mesophiles.

Methanogens in extreme environments

In deep basaltic rocks near the mid-ocean ridges, methanogens can obtain their hydrogen from the serpentinization reaction of olivine as observed in the hydrothermal field of Lost City. The thermal breakdown of water and water radiolysis are other possible sources of hydrogen. Methanogens are key agents of remineralization of organic carbon in continental margin sediments and other aquatic sediments with high rates of sedimentation and high sediment organic matter. Under the correct conditions of pressure and temperature, biogenic methane can accumulate in massive deposits of methane clathrates that account for a significant fraction of organic carbon in continental margin sediments and represent a key reservoir of a potent greenhouse gas.
Methanogens have been found in several extreme environments on Earth – buried under kilometres of ice in Greenland and living in hot, dry desert soil. They are known to be the most common archaea in deep subterranean habitats. Live microbes making methane were found in a glacial ice core sample retrieved from about three kilometres under Greenland by researchers from the University of California, Berkeley. They also found a constant metabolism able to repair macromolecular damage, at temperatures of 145 to –40 °C.
Another study has also discovered methanogens in a harsh environment on Earth. Researchers studied dozens of soil and vapour samples from five different desert environments in Utah, Idaho and California in the United States, and in Canada and Chile. Of these, five soil samples and three vapour samples from the vicinity of the Mars Desert Research Station in Utah were found to have signs of viable methanogens.
Some scientists have proposed that the presence of methane in the Martian atmosphere may be indicative of native methanogens on that planet. In June 2019, NASA's Curiosity rover detected methane, commonly generated by underground microbes such as methanogens, which signals possibility of life on Mars.
Closely related to the methanogens are the anaerobic methane oxidizers, which utilize methane as a substrate in conjunction with the reduction of sulfate and nitrate. Most methanogens are autotrophic producers, but those that oxidize CH₃COO⁻ are classed as heterotrophs instead.

Methanogens in the digestive tract of animals

The digestive tract of animals is characterized by a nutrient-rich and predominantly anaerobic environment, making it an ideal habitat for many microbes, including methanogens. Despite this, methanogens and archaea, in general, were largely overlooked as part of the gut microbiota until recently. However, they play a crucial role in maintaining gut balance by utilizing end products of bacterial fermentation, such as H₂, acetate, methanol, and methylamines.
Methanobrevibacter smithii is the predominant methanogenic archaeon in the microbiota of the human gut. Recent extensive surveys of archaea presence in the animal gut, based on 16S rRNA analysis, have provided a comprehensive view of archaea diversity and abundance. These studies revealed that only a few archaeal lineages are present, with the majority being methanogens, while non-methanogenic archaea are rare and not abundant. Taxonomic classification of archaeal diversity identified that representatives of only three phyla are present in the digestive tracts of animals: Methanobacteriota, Thermoplasmatota, and Halobacteriota. However, not all families and genera within these orders were detected in animal guts, but only a few genera, suggesting their specific adaptations to the gut environment.

Comparative genomics and molecular signatures

Comparative proteomic analysis has led to the identification of 31 signature proteins which are specific for methanogens. Most of these proteins are related to methanogenesis, and they could serve as potential molecular markers for methanogens. Additionally, 10 proteins found in all methanogens, which are shared by Archaeoglobus, suggest that these two groups are related. In phylogenetic trees, methanogens are not monophyletic and they are generally split into three clades. Hence, the unique shared presence of large numbers of proteins by all methanogens could be due to lateral gene transfers. Additionally, more recent novel proteins associated with sulfide trafficking have been linked to methanogen archaea. More proteomic analysis is needed to further differentiate specific genera within the methanogen class and reveal novel pathways for methanogenic metabolism.
Modern DNA or RNA sequencing approaches has elucidated several genomic markers specific to several groups of methanogens. One such finding isolated nine methanogens from genus Methanoculleus and found that there were at least 2 trehalose synthases genes that were found in all nine genomes. Thus far, the gene has been observed only in this genus, therefore it can be used as a marker to identify the archaea Methanoculleus. As sequencing techniques progress and databases become populated with an abundance of genomic data, a greater number of strains and traits can be identified, but many genera have remained understudied. For example, halophilic methanogens are potentially important microbes for carbon cycling in coastal wetland ecosystems but seem to be greatly understudied. One recent publication isolated a novel strain from genus Methanohalophilus which resides in sulfide-rich seawater. Interestingly, they have isolated several portions of this strain's genome that are different from other isolated strains of this genus. Some differences include a highly conserved genome, sulfur and glycogen metabolisms and viral resistance. Genomic markers consistent with the microbes environment have been observed in many other cases. One such study found that methane producing archaea found in hydraulic fracturing zones had genomes which varied with vertical depth. Subsurface and surface genomes varied along with the constraints found in individual depth zones, though fine-scale diversity was also found in this study. Genomic markers pointing at environmentally relevant factors are often non-exclusive. A survey of Methanogenic Thermoplasmata has found these organisms in human and animal intestinal tracts. This novel species was also found in other methanogenic environments such as wetland soils, though the group isolated in the wetlands did tend to have a larger number of genes encoding for anti-oxidation enzymes that were not present in the same group isolated in the human and animal intestinal tract. A common issue with identifying and discovering novel species of methanogens is that sometimes the genomic differences can be quite small, yet the research group decides they are different enough to separate into individual species. One study took a group of Methanocellales and ran a comparative genomic study. The three strains were originally considered identical, but a detailed approach to genomic isolation showed differences among their previously considered identical genomes. Differences were seen in gene copy number and there was also metabolic diversity associated with the genomic information.
Genomic signatures not only allow one to mark unique methanogens and genes relevant to environmental conditions; it has also led to a better understanding of the evolution of these archaea. Some methanogens must actively mitigate against oxic environments. Functional genes involved with the production of antioxidants have been found in methanogens, and some specific groups tend to have an enrichment of this genomic feature. Methanogens containing a genome with enriched antioxidant properties may provide evidence that this genomic addition may have occurred during the Great Oxygenation Event. In another study, three strains from the lineage Thermoplasmatales isolated from animal gastro-intestinal tracts revealed evolutionary differences. The eukaryotic-like histone gene which is present in most methanogen genomes was not present, alluding to evidence that an ancestral branch was lost within Thermoplasmatales and related lineages. Furthermore, the group Methanomassiliicoccus has a genome which appears to have lost many common genes coding for the first several steps of methanogenesis. These genes appear to have been replaced by genes coding for a novel methylated methogenic pathway. This pathway has been reported in several types of environments, pointing to non-environment specific evolution, and may point to an ancestral deviation.