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1 Introduction

Methanogens are microorganisms that produce methane as the end-product of their anaerobic respiration. All methanogens share three common features. (1) They are obligate methane producers, obtaining all or most of their energy for growth from producing large quantities of methane. (2) They are archaea, belonging to the phylum Euryarchaeota. (3) They are strict anaerobes, limiting their growth to anaerobic environments.

Methanogens can only utilize a restricted number of substrates for methane production or methanogenesis. The substrates are limited to three major types: CO2 + H2 or a few other electron donors such as formate, methyl-group-containing compounds, and acetate. Methanogens using these three types of substrates are classified as hydrogenotrophs, methylotrophs, and acetotrophs, respectively. Most organic substances, for instance, carbohydrates, proteins, and long-chain fatty acids and alcohols, are not substrates for methanogenesis. Exceptions are that some hydrogenotrophs can also use secondary alcohols, such as 2-propanol, 2-butanol, and cyclopentanol, as electron donors. A small number can use ethanol (Bleicher et al., 1989; Frimmer and Widdel, 1989; Widdel, 1986; Widdel et al., 1988). However, even these organic compounds, which can obviously be assimilated, are only incompletely oxidized to ketones (secondary alcohols) and acetate (ethanol), and methane is derived from CO2 reduction.

Methanogenesis is a complex process that requires a number of unique enzyme complexes and unusual coenzymes (reviewed by Hedderich and Whitman, 2006). Although the methanogenesis pathways of the three nutritional groups start differently, the final steps leading to methane are common in virtually all methanogens. The bioenergetics of methanogenesis employs both proton and sodium gradients generated by primary pumps for ATP synthesis. Due to the complexity of methanogenesis, all common methanogens are expected to originate from an ancient ancestor.

2 Taxonomy of Methanogens

Although methanogens are united by a few common features, they are phylogenetically diverse. The taxonomy of methanogens that has been developed in the last 3 decades has aimed to reflect the phylogenetic diversity of methanogens and be consistent with the taxonomy of other prokaryotes (Balch et al., 1979; Boone et al., 1993; Whitman et al., 2001). An overview of the current taxonomy of methanogens is given in Table 1 . Organisms from different orders have less than 82% 16S rRNA sequence similarity. Organisms with less than 88–93% and less than 93–95% 16S rRNA sequence similarity are separated into different families and genera, respectively. Organisms are distinguished as separate species if their DNA reassociation is less than 70%, the change in the melting temperature of their hybrid DNA is greater than 5°C, and substantial phenotypic differences exist (Stackebrandt et al., 2002; Wayne et al., 1987). When 16S rRNA data are available, organisms with a similarity of less than 98% are considered as separate species. However, sequence similarity of greater than 98% is not considered as sufficient evidence that two organisms belong to the same species.

Table 1 Taxonomy of methanogens
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Methanogens are currently classified into five orders: Methanobacteriales, Methanococcales, Methanomicrobiales, Methanosarcinales, and Methanopyrales (Whitman et al., 2001, 2006). This taxonomy is supported by comparative 16S rRNA gene sequence analyses as well as distinctive phenotypic properties, such as different cell envelope structure, lipid composition, and substrate range. Some representative characteristics are listed in Table 2 and further described in following chapters.

Table 2 Some characteristics of the methanogen orders
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A novel order of methanogen, Methanocellales, was proposed recently (Sakai et al., 2008). This order is currently represented by a single strain, Methanocella paludicola strain SANAE, which was originally isolated from rice paddy soil (Sakai et al., 2007). Based on comparative 16S rRNA gene sequence analysis, strain SANAE is affiliated with Rice Cluster I (RC-I), a clone lineage within the radiation of Methanosarcinales and Methanomicrobiales (review in Conrad et al., 2006). The 16S rRNA gene sequence similarities between strain SANAE and members of Methanosarcinales range from 80% to 82.8%, and those between the strain and members of Methanomicrobiales range from 77.5% to 82.4% (Sakai et al., 2008). This novel strain utilizes H2 + CO2 or formate as methanogenesis substrates. The cells are nonmotile and rod shaped. The growth temperature ranges from 25°C to 40°C, with an optimum of 35–37°C. The pH for growth ranges from 6.5 to 7.8, with an optimum of 7.0. The salinity for growth ranges from 0% to 0.1% (w/v) NaCl. It is physiologically distinguished from members of Methanosarcinales, which can use acetate and methylated compounds for methanogenesis. However, the phenotypic distinction between strain SANAE and members of Methanomicrobiales needs further investigation.

3 Phylogeny of Methanogens

All modern methanogens share the same set of homologous enzymes and cofactors required for methanogenesis, suggesting an ancient monophyletic origin of methanogens. In the phylogenetic tree based on 16S rRNA gene sequences, methanogens are separated into two major groups (Fig. 1 ). The Class I methanogens include Methanobacteriales, Methanococcales, and Methanopyrales, and the Class II methanogens include Methanomicrobiales and Methanosarcinales (Bapteste et al., 2005). Non-methanogenic lineages, Archaeoglobales, Halobacteriales, and Thermoplasmatales, are interspersed in the tree. Three hypotheses are proposed to explain this branching of methanogens.

  1. 1.

    Methanogens and these non-methanogen lineages shared a common ancestor, and genes required for methanogenesis were lost in these non-methanogens. This hypothesis is supported by the presence of a few genes encoding methanogenesis enzymes in the genome of Archaeoglobus fulgidus but is challenged by aerobic growth in both the Halobacteriales and Thermoplasmatales. This hypothesis also suggests that the common ancestor of Euryarchaeota was a methanogen (Gribaldo and Brochier-Armanet, 2006).

    Figure 1
    figure 1_42

    Phylogenetic tree for the methanogenic archaea and other euryarchaeotes based upon 16S rRNA sequences. The alignment was manually edited to include 1,251 positions. The tree was constructed with the neighbor-joining algorithm in MEGA4. Bootstrap analysis was performed with 1,000 replicates, and values greater than 50% are labeled on the nodes. The scale bar is 0.05 expected nucleotide substitutions per site. The 16S rRNA sequence of Methanosalsum zhilinae was according to Mathrani et al. (1988). The GenBank accession numbers for the other sequences are given following their names.

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  2. 2.

    Methanogenesis in various branches was acquired by horizontal gene transfer (HGT). However, the core genes required for methanogenesis are not linked on the genomes of methanogens, thus the simultaneous acquisition via lateral transfer is unlikely, and the transfer of single genes would not confer a selective advantage (Gribaldo and Brochier-Armanet, 2006).

  3. 3.

    The phylogeny based on 16S rRNA gene is misleading, and methanogens and Archaeoglobus shared a common ancestor exclusive of all other archaea. This hypothesis is supported by a recent phylogenomics analysis showing that ten proteins are exclusively shared in methanogens and A. fulgidus (Gao and Gupta, 2007), while no proteins are exclusively shared in methanogens and any of the Halobacteriales or Thermoplasmatales (Gao and Gupta, 2007). Therefore, methanogens and Archaeoglobus appear to have a closer relationship within the Euryarchaeota, but this leaves open the question of the apparently paradoxical phylogenetic position of both the Halobacteriales and Thermplasmatales. Therefore, more genomic sequences of archaea are needed to prove this hypothesis.

4 Ecology of Methanogens

Methanogens are abundant in a wide variety of anaerobic habitats such as marine sediments, freshwater sediments, flooded soils, human and animal gastrointestinal tracts, anaerobic digestors, landfills, and geothermal systems (Liu and Whitman, 2008). In some natural habitats, methanogens are also present in micro-oxic environments. For example, members of Methanobrevibacter have been isolated from large dental caries and subgingival plaque in the human mouth and gut periphery in termites. They are also somewhat oxygen tolerant, probably due to the presence of catalase activity and the protection given by O2-utilizing microbes (Brusa et al., 1987; Belay et al., 1988; Leadbetter and Breznak, 1996). RC-I methanogens are prevalent in rice rhizosphere, which is transiently oxic, and a reconstituted RC-I genome encodes a unique set of antioxidant enzymes, which may explain an aerotolerant life style (Erkel et al., 2006).

In methanogenic habitats, electron acceptors such as \({\rm O_2, \rm NO_3^-,\rm Fe^{3+}, \ and \ {SO_4^2{^-}}}\) are limiting. When electron acceptors other than CO2 are present, methanogens are outcompeted by the bacteria that utilize them. This phenomenon occurs mainly because the reduction of these compounds is thermodynamically more favorable than CO2 reduction to methane. However, because CO2 is generated during fermentations, it is seldom limiting in anaerobic environments. Besides methanogens, homoacetogens are another group of anaerobes that can reduce CO2 for energy production. However, acetogenesis with H2 is thermodynamically less favorable than methanogenesis. Therefore, homoacetogens do not compete well with methanogens in many habitats. However, homoacetogens outcompete methanogens in some environments, such as the hindgut of certain termites and cockroaches. Possible explanations may be their metabolic versatility as well as lower sensitivity to O2.

5 Research Needs

Recent culture-independent studies have revealed the presence of novel phylogenetic groups of methanogens. Their isolation and characterization will shed new insight into these organisms. For instance, investigations of rumen methanogens have found a novel lineage containing at least two families. The 16S rRNA gene sequences of this group have similarities closest to, but less than 80%, with those of Methanosarcinales (Nicholson et al., 2007). The Rice Cluster I is abundant in rice paddy soils, but only one strain has been isolated so far. Discovery and isolation of new strains will certainly add to our knowledge of the diversity of methanogens.

Methanogens have fewer easily determined physiological characteristics than most bacteria. Comparative 16S rRNA gene sequence analyses are indispensable for determination of taxonomic levels higher than species. However, it is frequently insufficient for taxonomy of methanogens at species and subspecies levels. For instance, some isolates of Methanobrevibacter have >98% 16S rRNA gene sequence similarities but exhibit less than 50% DNA relatedness, suggesting that they belong to different species (Lin and Miller, 1998; Keswani and Whitman, 2001). The discovery of novel molecular markers is desirable. The methyl-coenzyme M reductase alpha-subunit (mcrA) gene has been applied as a phylogenetic marker for methanogens in addition to 16S rRNA genes (Springer et al., 1995) and as a target for the detection of methanogens in a wide range of environments (Ohkuma et al., 1995; Lueders et al., 2001; Luton et al., 2002; Earl et al., 2003; Kemnitz et al., 2004). Phylogenomic analyses based upon whole-genome sequences may lead to improvement of the taxonomy and better view of phylogenetic relationships. A more complete database of methanogen genome sequences is required for this purpose.