1 Introduction

Methanogens are microorganisms that produce methane as the end-product of their anaerobic respiration. All methanogens share three common features. (i) They are obligate methane producers, obtaining all or most of their energy for growth from producing large quantities of methane. (ii) They are archaea, belonging to the phylum Euryarchaeota and possibly other archaeal phyla too. (iii) They are obligate anaerobes, limiting their growth to anaerobic environments.

Then known 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 (Widdel 1986; Widdel et al. 1988; Bleicher et al. 1989; Frimmer and Widdel 1989). Athough these organic compounds can obviously be assimilated, they 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 in 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 modern methanogens perhaps originate from a common ancient ancestor.

2 Taxonomy and Phylogeny 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 three 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. 1993b; Whitman et al. 2001b). 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 (Wayne et al. 1987; Stackebrandt et al. 2002). 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 a sufficient evidence that two organisms belong to the same species.

Table 1 Taxonomy of methanogens (Modified from Liu (2010e))
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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 seven orders (Fig. 1). Non-methanogenic lineages such as Archaeoglobales and Thermoplasmatales, are interspersed in the tree. Phylogenomic studies using more gene markers including ribosomal proteins and/or methanogenesis proteins further classified methanogens collectively into three classes (Bapteste et al. 2005; Anderson et al. 2009). The Class I methanogens include Methanobacteriales , Methanococcales , and Methanopyrales , the Class II methanogens include Methanomicrobiales , and the Class III methanogens include Methanosarcinales. However, when Methanocellales was included in phylogenomic analyses, the boundaries between the Classes II and III could not be fully resolved, suggesting that they could also belong to a single class (Lyu and Lu 2017). Although the seventh order Methanomassiliicoccales is distantly related to all three methanogen classes, its close affiliation to the Class Thermoplasmata could not warrant an immediate establishment of a fourth methanogen class.

Fig. 1
figure 1

Maximum-likelihood tree based on nearly full length 16S rRNA gene sequences from type species of 34 methanogen genera. The tree was built by FastTree 2.1.5 using Thermococcus celer as an outgroup. Bootstrap values >0.77 are indicated at nodes and were based on 1000 replicates (Price 2010). There were a total of 1555 positions in the final dataset, which were aligned in the RDP 11 database. The scale bar represents substitutions per position. The GenBank accesion numbers are indicated following the species name

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Four hypotheses are proposed to explain the branching of methanogens. (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). However, this view is now challenged by the possible presence of methanogens outside Euryarchaeota as shown by metagenomic surveys (Evans et al. 2015; Vanwonterghem et al. 2016). (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) 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 phylogenomics analyses showing that 10 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. However, the presence of methanogens in the Thermoplasmata suggests otherwise. (4) The last archaeal common ancestor was a methanogen, and the methanogenesis pathway was inherited, modified or lost in various lineages throughout evolution. This view is supported by (i) recent metagenomics surveys that indicate possible presence of methanogens in at least two other archaeal phyla besides the Euryarchaeota (Evans et al. 2015; Vanwonterghem et al. 2016), and (ii) the root of the archaeal tree based on phylogenomic analyses was placed between Euryarchaeota and the rest of archaeal phyla (Petitjean et al. 2015).

Methanogens are currently classified into seven orders: Methanobacteriales, Methanococcales, Methanomicrobiales, Methanosarcinales, Methanomassiliicoccales, Methanocellales and Methanopyrales (Whitman et al. 2001b, 2006; Sakai et al. 2008; Iino et al. 2013). This taxonomy is supported by comparative 16S rRNA gene sequence and phylogenomic analyses as well as distinctive phenotypic properties, such as different cell envelope structures, lipid compositions, and substrate ranges. Some representative characteristics are listed in Table 2 and further described in following subsections.

Table 2 Some characteristics of the methanogen orders (Modified from Liu (2010e))
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2.1 Methanobacteriales

Methanobacteriales are currently classified into two families and five genera based upon 16S rRNA sequences, DNA reassociation levels, and phenotypic characteristics. The two families Methanobacteriaceae and Methanothermaceae are distinguished by 16S rRNA sequence similarities below 89% and differences in cell wall structure and growth temperatures. The family Methanobacteriaceae contains three mesophilic genera – Methanobacterium, Methanobrevibacter, and Methanosphaera – and one thermophilic genus Methanothermobacter. Members of the Methanobacteriaceae possess pseudomurein as a major component of the cellular envelope. The family Methanothermaceae is represented by one hyperthermophilic genus, Methanothermus. Members of the Methanothermaceae possess a protein surface layer in addition to the pseudomurein layer.

The placement of the hyperthermophilic Methanothermus into a separate family from other Methanobacteriales genera is justified by the deep branching of the phylogeny of its 16S rRNA gene (Schuchmann and Muller 2014). The 16S rRNA gene sequence similarities within the Methanothermus species are much higher (98%) than the similarities between Methanothermus and other members of the Methanobacteriales (83–89%). This classification is further confirmed by DNA reassociation. For instance, the DNA relatedness between Methanothermus isolates and Methanothermobacter thermoautotrophicus strain IM is 2–8% (Lauerer et al. 1986). Phenotypically, the genus Methanothermus is distinguished from other Methanobacteriales by their high temperature optima (80–88 °C), double-layered cell wall, and motility by bipolar polytrichous flagellation.

Methanobacteriaceae is a diverse family, including mesophilic and thermophilic species. The phylogeny of the 16S rRNA gene indicates that the thermophilic species are divergent from mesophilic members at the genus level. The 16S rRNA sequence similarities within the thermophilic genus Methanothermobacter are above 98%, while the similarities between thermophilic and mesophilic members of Methanobacteriaceae are generally below 93% (Wasserfallen et al. 2000). The DNA relatedness between Methanothermobacter species are 22–47%, confirming that they are genetically distant and should be assigned to separate species (Boone et al. 2001a).

The separation of mesophilic members of Methanobacteriales into three genera is supported by both genetic and phenotypic analyses. Species of Methanobacterium are usually autotrophs, while species of Methanobrevibacter and Methanosphaera are commonly mixotrophic or heterotrophic. Species of Methanosphaera use only H2 and methanol as substrates for methanogenesis, while all species of Methanobrevibacter and Methanobacterium can use H2 and CO2.

Members of the order Methanobacteriales use a limited range of substrates for methanogenesis. Most of them reduce CO2 to CH4 with H2. Some Methanobacterium species can also reduce methanol with H2, which are the exclusive substrates for the genus Methanosphaera. There is one Methanobacterium species that can also reduce methylamine with H2. Some Methanobacteriales members can also use formate, CO, or secondary alcohols as electron donors. Some species can grow autotrophically using CO2 as the sole carbon source, and some species are mixotrophs or heterotrophs, which may require acetate, amino acids, peptones, yeast extract, vitamins, and/or rumen fluid for growth. Ammonium is a major nitrogen source. Sulfide can serve as the sole sulfur source, and some species can reduce elemental sulfur to sulfide. Cells are generally rod-shaped with a length of 0.6–25 μm, often forming chains or filaments up to 40 μm in length. Cells typically stain Gram positive, but the wall does not contain muramic acid. Pesudomurein is the predominant polymer in the cell wall. Members of the genus Methanothermus have double-layered cell wall, consisting of an inner pseudomurein layer and an outer S-layer composed of protein. The cellular lipids contain caldarchaeol, archaeol, and, in some species, hydoxyarchaeol as core lipids. The polar lipids can contain glucose, N-acetylglucosamine, myo-inositol, ethanolamine, and serine, depending on the species. Most species are nonmotile. However, Methanobacterium movens and members of the genus Methanothermus are motile via one or two polar flagella and peritrichous flagella, respectively. The optimum growth temperatures of members of the Methanobacteriales vary from 20 °C to 88 °C. The genus Methanothermus can grow at temperatures up to 97 °C, while multiple Methanobacterium species can grow at as low as 10 °C and one species can even grow at 0 °C. The pH optima of Methanobacteriales members vary from 5.5 to 9.

Descriptive properties of the Methanobacteriales are summarized in Tables 3, 4, 5, 6, and 7. Further information can be found in Bonin and Boone (2006) and Boone et al. (2001a). Our current knowledge on the diversity of the Methanobacteriales is largely incomplete. As an example, investigations of 16S rRNA gene from clone libraries recognized a large number of uncultured Methanobrevibacter, especially from the rumen and termite gut (Dighe et al. 2004; Wright et al. 2004). Moreover, the cloned sequences from termite gut formed separate lineages from cultured Methanobrevibacter (Dighe et al. 2004). The correlation between ecological habitat and 16S rRNA based phylogeny need more ecological surveys to unravel.

Table 3 Descriptive characteristics of the species of the genus Methanobacterium (Modified from Liu (2010a))
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Table 4 Descriptive characteristics of the species of the genus Methanobrevibacter (Modified from Liu (2010a))
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Table 5 “Descriptive characteristics of the species of the genus Methanosphaera ” (Originally published in Liu (2010a), published with kind permission of © Springer Science+Business Media New York, 2003. All rights reserved)
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Table 6 Descriptive characteristics of the species of the genus Methanothermobacter (Modified from Liu (2010a))
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Table 7 “Descriptive characteristics of the species of the genus Methanothermus” (Originally published in Liu (2010a), published with kind permission of © Springer Science+Business Media New York, 2003. All rights reserved)
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2.2 Methanococcales

The order Methanococcales is composed of two families, Methanocaldococcaceae and Methanococcaceae , which are distinguished by 16S rRNA sequence similarities below 93% and differences in growth temperatures. The Methanocaldococcaceae are all hyperthermophilic , while the Methanococcaceae are extremely thermophilic and mesophilic. Members of this order are all capable of forming methane by CO2 reduction with H2. Many species can use formate as an alternative electron donor. Most species can grow autotrophically.

Phylogenetic analyses with DNA sequences reveal a high diversity of the Methanococcales. The sequence similarities of the 16S rRNA genes between hyperthermophilic and mesophilic methanococci are generally below 90%. For instance, the 16S rRNA gene sequence similarity between the mesophile Methanococcus voltae and the hyperthermophile Methanocaldococcus infernus is about 85%, which is comparable to the similarity between Escherichia and Pseudomonas. In addition, the mesophilic methanococci possess 91–96% (average 94%) 16S rRNA gene sequence similarities and 5–30% DNA reassociation values, suggesting that they are related only at the genus level (Keswani et al. 1996).

The Methanococcales are currently divided into two families and four genera, according to their growth temperatures. The family Methanocaldococcaceae includes two hyperthermophilic genera, Methanocaldococcus and Methanotorris. The family Methanococcaceae includes the mesophilic genus Methanococcus and the extremely thermophilic genus Methanothermococcus. This taxonomy generally agrees with the phylogeny of the 16S rRNA genes (Liu 2010b), in which the lineages formed by the deepest bifurcation represent the two methanococcal families. However, some ambiguity remains. For instance, 16S rRNA gene sequences indicate that Methanococcus aeolicus forms a deep branch of the mesophilic methanococci and is more closely related to the thermophile Methanothermococcus okinawensis (95% sequence similarity) than to the other Methanococcus (91–93% sequence similarity). In addition, Methanothermococcus okinawensis also has low sequence similarity to the other thermophile Methanothermococcus thermolithotrophicus (95% sequence similarity). Therefore, the phylogenetic analysis implies that Methanococcus aeolicus and Methanothermococcus okinawensis could be classified into two novel genera. Nevertheless, phylogeny of additional genes and phenotypic differences other than growth temperature should be examined to justify reclassification.

DNA relatedness and cellular protein patterns are often determined for the phylogenetic and taxonomic analyses of methanococci. They are especially useful to distinguish relationships at the species and subspecies levels, at which levels the 16S rRNA gene sequence analysis is frequently incongruent. For instance, two heterotrophic Methanococcus voltae strains A2 and A3 exhibit 37% DNA relatedness to the type train PS (Keswani et al. 1996). Similarly, four autotrophic Methanococcus maripaludis strains C5, C6, C7, and C8 exhibit 54–69% DNA relatedness to the type strain JJ (Keswani et al. 1996). Moreover, differences in cellular protein patterns between these strains are also readily recognized. Therefore, classification of these strains into separate species is suggested based on their genetic diversities. However, because distinguishable phenotypic properties are few, these strains are not currently considered as novel species.

Autotrophy and thermophily are represented in both methanococcal families, suggesting that the mesophilic methanococci may have evolved from an autotrophic thermophile (Keswani et al. 1996). The heterotrophy of Methanococcus voltae is possibly a recently acquired characteristic. This hypothesis is consistent with the presence of enzymes required for autotrophic CO2 fixation in M. voltae (Shieh et al. 1988).

Members of the Methanococcales or the methanococci are coccoid methanogens isolated from marine environments. They share a set of phenotypic characteristics. They all use H2 or formate to reduce CO2 for methanogenesis. Acetate, methyl-containing compounds, and alcohols are not used as substrates for methanogenesis. Most of them can grow autotrophically with CO2 as the sole carbon source. Sulfide is a sufficient sulfur source for all methanococci, and elemental sulfur is reduced to sulfide with slight inhibition of growth in most strains. Ammonium is a sufficient nitrogen source for all methanococci, and nitrogen gas, nitrate, and alanine are used as a nitrogen source by some species. They all require sea salts for optimal growth. Cells are irregular cocci, 1–3 μm in diameter during balanced growth. Most of them are motile by means of polar tuft(s) of flagella. Cells strain Gram negative. They are susceptible to lysis by 0.01% (w/v) SDS and hypotonic solutions. Cell envelopes are composed of a protein cell wall or S-layer. Glycoproteins and cell wall carbohydrates are not abundant. The cellular lipids contain archaeol, caldarchaeol, hydroxyarchaeol, and macrocyclic archaeol, depending upon the species. The polar lipids can contain glucose, N-acetylglucosamine, serine, and ethanolamine. The optimal growth temperatures of methanococci are diverse, ranging from 35 °C to 88 °C. They are among the fastest growing methanogens at either mesophilic or thermophilic temperatures, with generation times of about 2 h at 37 °C and less than 30 min at 85 °C.

Descriptive properties of the methanococci are summarized in Tables 8 and 9. Further information can be found in Whitman et al. (2001a), and Whitman and Jeanthon (2006). Creation of new families and genera may be necessary with addition of new isolates and identification of new phenotypic and genetic markers. The Methanotorris may represent a new family because they have only 92–93% 16S rRNA similarities with the Methanocaldococcus. These two groups are also distinguished by the presence of hydroxyarchaeol and the absence of caldarchaeol in the Methanotorris. Methanococcus aeolicus and Methanothermococcus okinawensis may represent two new genera because they form a lineage separate from other Methanococcaceae in the 16S rRNA phylogenetic tree.

Table 8 Descriptive characteristics of the species of the genera Methanocaldococcus and Methanotorris (Modified from Liu (2010b))
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Table 9 “Descriptive characteristics of the species of the genera Methanococcus and Methanothermococcus ” (Originally published in Liu (2010b), published with kind permission of © Springer Science+Business Media New York, 2003. All rights reserved)
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2.3 Methanomicrobiales

The order Methanomicrobiales is composed of four families, Methanomicrobiaceae , Methanocorpusculaceae , Methanospirillaceae , and Methanoregulaceae , which are distinguished by 16S rRNA sequence similarities below 89%. The Methanospirillaceae is further distinguished from the other two families by its unique morphology of curved rod-shape and exterior sheath. All members of this order are capable to produce methane by CO2 reduction with H2. Formate and secondary alcohols are used as alternative electron donors in many species.

Because the members of Methanomicrobiales share many phenotypic characteristics, it is difficult to divide them based solely on their physiological properties. Both of the families Methanomicrobiaceae and Methanocorpusculaceae contain coccoid organisms, and nearly all members require organic carbon sources for growth (except Methanofollis aquaemaris). Therefore, they are difficult to distinguish except by molecular phylogenetic analyses. The family Methanospirillaceae is distinguished from the other three families by its unique morphology of curved rod-shape and capability of autotrophic growth. The family Methanoregulaceae is unique by having members that grow in acidic conditions.

The family Methanomicrobiaceae is divided into six genera. The 16S rRNA gene sequence similarities between different genera are 87–95%, suggesting that they are sufficiently distinctive at genus level. The 16S rRNA gene sequence similarities between different species within a genus are above 95.4%. Both Methanomicrobium and Methanolacinia are represented by a single species. Cells of both genera are rod-shaped, but they can be differentiated by some other physiological characters. In addition to H2, Methanolacinia paynteri can use secondary alcohols to reduce CO2. In contrast, Methanomicrobium mobile can only use H2 or formate as electron donors for methanogenesis. Methanolacinia paynteri is a marine organism, while Methanomicrobium mobile was isolated from bovine rumen. Cells of Methanoculleus, Methanofollis, and Methanogenium are irregular cocci. These three genera are difficult to differentiate by phenotypic characteristics. Methanoplanus differs from the other genera by its plate or disc cell shape.

The family Methanospirillaceae is represented by a single species, Methanospirillum hungatei. Cells have a unique spiral shape that is not found in other methanogens. Cell walls consist of an inner protein S-layer and a rigid paracrystalline outer sheath conferring the α-helical spiral shape of the cells (Sprott and McKellar 1980; Sprott et al. 1983). Cells usually grow as single cells or short filaments within their sheath. The cellular lipid of M. hungatei contains two unusual phosphoglycolipids, which are derivatives of the dibiphytanyl diglycerol tetraether. One of the free hydroxyls of this tetraether is esterified with glycerophosphoric acid, and the other is linked to a disaccharide (Kushwaha et al. 1981).

The family Methanocorpusculaceae is represented by the genus Methanocorpusculum. Cells are irregular cocci with diameters generally <1 μm. All species can use formate in addition to H2 as electron donor for methanogenesis. For some species, secondary alcohols are alternative electron donors. Acetate and either yeast extract, peptones, or rumen fluid are required as carbon sources. The habitats of Methanocorpusculum are usually anaerobic digesters or freshwater sediments. They have not been found in marine environments.

The family Methanoregulaceae is divided into three genera (Sakai et al. 2012). The 16S rRNA gene sequence similarities between different genera are 93–96%, suggesting that they are sufficiently distinctive at genus level. Both Methanolinea (Imachi et al. 2008; Sakai et al. 2012) and Methanoregula (Brauer et al. 2006; Wang et al. 2009) are represented by two species, while Methanosphaerula is represented by one (Cadillo-Quiroz et al. 2009). Methanolinea is morphologically distinct from other Methanomicrobiales by forming rod-shaped, multicellular filaments within a sheath-like structure. Methanoregula and Methanosphaerula are distinguished from others by their acidophilic growth.

The assignment of Methanocalculus into a novel family is tentative. The 16S rRNA sequence similarities between all known Methanocalculus species are >98%, but those between Methanocalculus and other methanogens are <91%. Different species of Methanocalculus exhibited <10–51% DNA relatedness. The closest neighbor of Methanocalculus in the phylogenic tree based on 16S rRNA gene is Methanocorpusculum. All members of Methanocalculus are irregular cocci, can only use H2 and CO2 or formate for methanogenesis, and require acetate for growth.

All members of the order Methanomicrobiales produce methane using CO2 as the electron acceptor and H2 as the electron donor. Most species use formate and many species also use secondary alcohols as alternative electron donors, while two unique species can also grow on primary alcohols. They cannot use acetate and methyl-group containing compounds for methanogenesis. Most species are mixotrophic and require acetate as a carbon source; some species also require additional organic growth factors. Their morphologies are diverse, including cocci, rods, and sheathed rods. Most cells have single-layered protein cell walls, but cells of Methanospirillum hungatei are surrounded by an external sheath. Peptidoglycan and pseudomurein are absent. The cellular lipids contain archaeol and caldarchaeol as core lipids. Hydroxyarchaeol is absent. Glucose, galactose, aminopentanetetrols, and glycerol are common polar lipids; and aminopentanetetrols are unique to this order of organisms. Motility varies between species. Most species are mesophilic , with the exceptions of two psychrophilic species (Methanogenium marinum and Methanogenium frigidum) and one thermophilic species (Methanoculleus thermophilicus). Most species grow best near neutral pH. Exceptions are Methanoregula boonei and Methanosphaerula palustris, which have an optimal pH of 5.1~5.7 and were isolated from acidic peat bog; and Methanocalculus alkaliphilus and Methanocalculus natronophilus, which grow best at pH of 9.5 and were isolated from soda lake sediments. Many species are marine organisms and grow optimally with 0.1–1 M of NaCl. Descriptive properties of the Methanomicrobiales are summarized in Table 10. Further information can be found in Boone et al. (2001b) and Garcia et al. (2006).

Table 10 Descriptive characteristics of the species of the order Methanomicrobiales (Modified from Liu (2010c))
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2.4 Methanosarcinales

The order Methanosarcinales is divided into three families, Methanosarcinaceae , Methanosaetaceae and Methermicoccus based on phenotypic properties and 16S rRNA gene sequence analysis (Cheng et al. 2007). The three families are distinguished by 16S rRNA sequence similarities below 91% and differences in substrates for methanogenesis, lipid components, and cell wall structures. The Methanosarcinaceae are all capable of producing methane from methyl group containing compounds, and some can use acetate or H2/CO2. The cells can form aggregates within an outer layer composed of heteropolysaccharide. The Methanosaetaceae can only produce methane by splitting acetate. The cells can form chains within a proteinaceous sheath. The family Methermicoccus is represented by only one species, which is a thermophilic, methylotrophic methanogen isolated from an oilfield (Cheng et al. 2007).

The family Methanosarcinaceae currently comprises eight genera, Methanococcoides, Methanohalobium, Methanohalophilus, Methanolobus, Methanomethylovorans, Methanosalsum, Methanimicrococcus and Methanosarcina. The genus Methanosarcina can be differentiated from other genera by the unique morphology of pseudosarcinae or large cysts, which are formed by aggregation of cells within a common outer layer. The outer layer is composed of heteropolysaccharide, consisting mainly of galactosamine, glucose, mannose, and galacturonic acid. Some Methanosarcina species can also be distinguished from other genera of Methanosarcinaceae by their ability to split acetate for methanogenesis. The genus Methanohalobium is represented by a single species, M. evestigatum, which is an extreme halophile that requires 4 M of NaCl for optimal growth. The genus Methanosalsum is represented by M. zhilinae and M. natronophilum, which are moderate halophiles and alkaliphiles. The genus Methanohalophilus comprises moderate halophilic and halotolerant species, which grow best with 1–2 M of NaCl. The genera Methanococcoides and Methanolobus are difficult to differentiate by phenotypic properties, as they all use methylated compounds for methanogenesis; they require phylogenetic analysis for taxonomy. The genus Methanimicrococcus is represented by a single spcies Methanimicrococcus blatticola, which is a dominant methylotrophic methanogen in the cockroach hindgut (Sprenger et al. 2000). It has 83.4–89.8% 16S rRNA gene sequence similarities with other species of Methanosarcinales, suggesting that it could potentially represent a new family. This is further supported by the fact that it cannot disproportionate methyl-group containing compounds, a feature shared by all other Methanosarcinaceae spp. Instead, methanol and methylated amines must be reduced with H2 for methanogenesis. This obligately hydrogenotrophic and methylotrophic mode of growth is shared with Methanosphaera and Methanomassiliicoccus, which belongs to the Methanobacteriales and Methanomassiliicoccales, respectively.

Members of the family Methanosaetaceae use acetate as the sole energy source. Acetate and CO2 serve as carbon sources. Cells form filament-like structures within the sheath, which is composed predominantly with proteins and contains carbohydrates.

Methanogens from only two genera, Methanosarcina and Methanosaeta, can use acetate as a substrate for methanogenesis. However, they metabolize acetate differently. Methanosarcina is a relative generalist that prefers methanol and methylamine to acetate, and many species also utilize H2. Methanosaeta is a specialist that uses only acetate. Methanosaeta is a superior acetate utilizer in that it can use acetate at concentrations as low as 5–20 μM, while Methanosarcina requires a minimum concentration of about 1 mM (Jetten et al. 1992). The difference of acetate affinity is probably due to different systems for acetate activation. Moreover, based upon their genome sequences, these two genera probably have different modes of electron transfer and energy conservation, even though the methanogenesis pathways are likely to be similar (Smith and Ingram-Smith 2007).

The family Methermicoccus is represented by Methermicoccus shengliensis. Its closest neighbor in the 16S rRNA phylogenetic tree is Methanosaeta (< 90.7% sequence similarities). It is morphologically differentiated from Methanosaeta by its coccoid-shape and formation of large cysts. Moreover, M. shengliensis uses methanol and methylated amines, but not acetate, for methanogenesis.

Members of the order Methanosarcinales have the widest substrate range among methanogens. All members can produce methane by disproportionating methyl-group containing compounds (methanol, methylamines, methylethanolamines, betaine, or methyl sulfides) or by splitting acetate. Some mesophilic Methanosarcia species can reduce CO2 with H2, but formate, secondary alcohols, and ethanol are not used as electron donors. Recently, it has been shown that Methermicoccus spp. are surprisingly capable of growth and methane production using methoxylated aromatic compounds (MACs) such as methoxy-benzoate (Mayumi et al. 2016). Ammonium and sulfide serve as the major nitrogen and sulfur sources, respectively. Their cellular morphologies are diverse, including cocci, pseudosarcinae, and sheathed rods. Most cells have protein cell walls, and some cells are surrounded by a sheath or acidic heteropolysaccharide. Most strains are nonmotile. The cellular lipids contain archaeol, hydroxyarchaeol, and caldarchaeol. Polar lipids can contain glucose, galactose, mannose, myo-inositol, ethanolamine, serine, and glycerol, depending upon the species. Most species of Methanosarcinales are mesophilic. Four species are moderately thermophilic (Methanosarcina thermophila, Methanomethylovorans thermophila, Methanosaeta thermophila, and Methermicoccus shengliensis), and six species are psychrotolerant (Methanococcoides alaskense, Methanococcoides burtonii, Methanosarcina lacustris, Methanosarcina soligelidi, Methanosarcina splelaei, and Methanosarcina baltica). Most species grow best at near neutral pH, except for three species that are alkaliphilic (Methanolobus oregonensis, Methanolobus taylorii, Methanosalsum natronophilum, and Methanosalsum zhilinae). Many species were isolated from marine environments and require a salinity near that of seawater for optimal growth. Some species are halophilic or halotolerant. Descriptive properties of members of the Methanosarcinales are summarized in Table 11. Further information can be found in Boone et al. (2001c) and Kendall and Boone (2006).

Table 11 Descriptive characteristics of the species of the order Methanosarcinales (Modified from Liu (2010d))
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2.5 Methanopyrales

The order of Methanopyrales is represented by only one species, Methanopyrus kandleri. It is hyperthermophilic and produces methane by CO2 reduction with H2. Genomic sequence analysis of M. kandleri suggests that it is closely related to Methanobacteriales and Methanococcales but possesses unusual features.

The phylogenetic position of M. kandleri is ambiguous. The phylogenic analyses based on 16S rRNA gene (Burggraf et al. 1991), elongation factor 1α (Rivera and Lake 1996), and transcription factors (Brochier et al. 2004) suggested that M. kandleri is distantly related to other methanogens and represent a separate lineage emerging at the base of the euryarchaeal phylum. On the other hand, phylogenetic analyses based on methyl coenzyme M reductase (MCR) operons (Nolling et al. 1996), translation factors (Brochier et al. 2004), and whole genome sequences (Slesarev et al. 2002; Gao and Gupta 2007) suggested that M. kandleri is more closely related to other methanogens and grouped with Methanobacteriales and Methanococcales. Indeed, M. kandleri encodes the core of proteins shared uniquely by methanogens such as proteins evolved in the methanogenesis pathway, and it closely resembles other methanogens in terms of local gene order. Therefore, M. kandleri very likely belongs to the monophyletic methanogen group and not a deep-branch close to the root of archaea. The deep branching in 16S rRNA phylogenetic tree is probably due to a very high GC content of M. kandleri, a characteristic shared by hyperthermophiles outside the methanogen group.

The genome of M. kandleri displays several unusual features (Slesarev et al. 2002; Brochier et al. 2004). The RNA polymerase subunit H is replaced by a homologous protein from a distantly related archael lineage. The transcription factor S (TFS) is missing. The diversity of predicted signal transduction systems and DNA-binding proteins are underrepresented. The histone protein is formed by a fusion of two monomers into a single peptide with two tandemly repeated histone folds. M. kandleri possesses a unique topoisomerase, Topo V, which is related to eukaryotic topoisomerase I (Slesarev et al. 1994). These unusual features suggest a high level of gene loss, gene capture, and gene fusion in this archaeon.

Methanopyrus kandleri is the only methanogen known so far that catalyzes methanogenesis at temperatures higher than 100 °C. It reduces CO2 with H2 for methanogenesis. It is an obligate chemolithoautotroph that uses CO2 as the sole carbon source. Ammonium and sulfide are the nitrogen and sulfur sources, respectively. The cells are rod-shaped and stain Gram positive. The cell wall is double layered. The inner layer is composed of a new type of pseudomurein, containing ornithine and lysine. The outer layer is detergent-sensitive, indicating a protein composition. The core lipid is composed of an unsaturated terpenoid lipid, which is considered the most primitive lipid in the evolution of membranes (Hafenbradl et al. 1993). The cells are motile via flagella arranged as polar tufts. They grow at temperatures ranging from 84 °C to 110 °C, with an optimum of 98 °C. The range of pH for growth is 5.5–7, with an optimum of 6.5. The optimal NaCl concentration for growth is 2.0% (w/v). The GC content of its DNA is 60 mol%. M. kandleri was isolated from hydrothermally heated deep-sea sediments and from a shallow marine hydrothermal system (Kurr et al. 1991).

2.6 Methanocellales

The order Methanocellales is represented by one family and genus, Methanocellaceae and Methanocella , respectively. Three species have been described, and they are distinguished by 16S rRNA sequence similarities below 92% and differences in growth temperatures, substrates for methanogenesis, possession of a flagellum , doubling time and NaCl range. The low 16S rRNA sequence similarities suggest potential separation into more genera, which is supported by comparative genomic studies (Sakai et al. 2011; Lyu and Lu 2015). The Methanocella are all capable of producing methane from H2/CO2, but acetate is required for growth. Formate can also be used as an alternative substrate by two species.

Members of Methanocellales are isolated from rice soils. They do not appear to grow autotrophically due to the requirement of acetate for growth. Sulfide and ammonium is a sufficient sulfur and nitrogen source, respectively. Cells are typically rods, but coccoid cells are also seen during late stage of growth. Cells can form a unique lens-shaped colony. Cell envelopes are composed of an S-layer as determined in Methanocella avoryzae. Cell envelopes have not been determined in Methanocella paludicola and Methanocella conradii, but they are resistant to lysis by 2.0% and 0.1% of SDS, respectively. A flagellum is also present in both M. avoryzae and M. conradii, but not in M. paludicola. Cellular lipids have not been determined. They all grow optimally in the absence of NaCl and at neutral pH. The optimal growth temperatures range from 37 °C to 55 °C. Descriptive properties of the Methanocellales are summarized in Table 12. Further information can be found in Sakai et al. (2008, 2010), and Lü and Lu (2012b).

Table 12 Descriptive characteristics of the species of the genus Methanocella
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2.7 Methanomassiliicoccales

The order Methanomassiliicoccales is represented by one family and genus, Methanomassiliicoccaceae and Methanomassiliicoccus, respectively (Dridi et al. 2012; Iino et al. 2013). Although a few enrichment cultures are available, only one species Methanomassiliicoccus luminyensis has been described (Borrel et al. 2012a, 2013; Dridi et al. 2012; Iino et al. 2013). This species was isolated from human faeces, and it reduces methanol with H2 to produce methane. However, genomic, transcriptomic and in vivo studies suggest that members of Methanomassiliicoccales also reduce tri-, di- and monomethylamine with H2 (Poulsen et al. 2013; Borrel et al. 2014; Brugere et al. 2014). Cells are non-motile cocci and lysed in 0.1% (w/v) SDS. It grows optimally at 1% of NaCl, 37 °C and at pH 7.6. Descriptive properties of the Methanomassiliicoccales are summarized in Table 13. Further information can be found in Dridi et al. (2012) and Brugere et al. (2014).

Table 13 Descriptive characteristics of Methanomassiliicoccus luminyensis
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2.8 Potential Novel Taxa

Through metagenomics guided discovery, a few potential novel taxa of methanogens have been proposed recently. That includes a euryarchaeon, Candidatus ‘Methanofastidiosa’, and members of the archaeal phyla Bathyarchaeota (previously known as the Miscellaneous Crenarchaeota Group) and Verstraetearchaeota previously represented by the Terrestrial Miscellaneous Crenarchaeota Group or TMCG) (Evans et al. 2015; Nobu et al. 2016; Vanwonterghem et al. 2016). They are all predicted to reduce different methylated compounds with H2 for methanogenesis, but members of Bathyarchaeota and Verstraetearchaeota may also use complex substrates such as lactate. Pure cultures are still needed to further confirm these findings, which would likely not only lead to proposals of novel methanogen classes but establishment of methanogen taxa outside the Euryarchaeota.

3 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). This cosmopolitan distribution of methanogens could be associated with their growth largely relied on only simple substrates such as H2/CO2, acetate, formate and other C1 compounds, which are widely available across ecosystems where complex substrates have to be degraded into simple substrates to drive the carbon cycle. A recent metagenomics survey has also predicted the presence of complex fermentation and β-oxidation pathways in the putative Bathyarchaeota methanogens, suggesting the ability of using complex substrates may be advantageous for methanogens that thrive in environments where degradation of complex substrates could be very slow (Evans et al. 2015). In addition, some methanogens as described in the taxonomy and phylogeny section can also survive extreme environmental conditions such as hyperthermophilic, psychrophilic , piezophilic , halophilic , alkaliphilic and acidophilic , which further expands their habitats.

In some natural habitats, methanogens are also present in microoxic 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 by O2-uptake aerobes (Brusa et al. 1987; Belay et al. 1988; Leadbetter and Breznak 1996). Methanocellales methanogens are prevalent in rice rhizosphere, which is transiently oxic, and their genomes encode a unique set of antioxidant enzymes, which may explain an aerotolerant life style (Erkel et al. 2006; Sakai et al. 2011; Lü and Lu 2012a; Lyu and Lu 2015, 2017).

In methanogenic habitats, electron acceptors such as O2, NO3−, Fe3+, and SO42− 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 reductions of these compounds are 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 are their metabolic versatility as well as lower sensitivity to O2. The ecology of each methanogen order is discussed below.

3.1 Methanobacteriales

Members of the Methanobacteriales are widely distributed in anaerobic habitats such as marine and freshwater sediments, soils, animal gastrointestinal tracts, anaerobic sewage digestors, and geothermal habitats. Methanobacterium has been cultivated from marine and freshwater sediments, groundwaters, soils, anaerobic digestors, and animal gastrointestinal tracts and has also been detected as endosymbionts in anaerobic ciliate (Embley et al. 1992). Methanobrevibacter has been isolated from rumens, feces, termite hindguts, human subgingival plaque, anaerobic digestors, and decaying wood tissues. Methanosphaera has only been isolated from animal gastrointestinal tracts but has been detected in anaerobic digestors (Weiss et al. 2008). Methanothermobacter has been cultivated from thermophilic anaerobic digestors and natural gas and oil fields (Nazina et al. 2006; Mochimaru et al. 2007). Methanothermus has only been isolated from solfarata hot springs.

3.2 Methanococcales

Members of the Methanococcales have all been isolated from marine environments. Methanococcus has been isolated from marine and salt marsh sediments. Methanothermococcus has been isolated from coastal geothermally heated sea sediments, deep sea hydrothermal vents, and reservoir water from marine oil fields (Nilsen and Torsvik 1996) and has been detected in continental high-temperature oil reservoirs (Orphan et al. 2000) and tropical hypersaline coastal lagoons (Clementino et al. 2008). Methanocaldococcus has only been isolated from deep sea hydrothermal vents. Methanotorris has been isolated from shallow and deep sea hydrothermal vents. Environmental 16S rRNA sequences closely related to Methanococcales have also been detected in anaerobic granular sludge (Liu et al. 2002; Diaz et al. 2003). Quantitative real-time PCR assays have also recently shown possible presence of Methanococcales in forest and grassland soils, but how specific the primers were remain unknown (Hofmann et al. 2016). Since this finding is very much unexpected, sequence data is also needed to make conclusive taxonomy inference.

3.3 Methanomicrobiales

Members of the Methanomicrobiales are widely distributed in anaerobic habitats, including marine and freshwater sediments, anaerobic sewage digestors, rice paddies, oil fields, groundwaters, and animal gastrointestinal tracts. Anaerobic digestors and sewage sludge are common habitats of Methanoculleus, Methanofollis, Methanocorpusculum, Methanospirillum, and Methanomicrobium. From marine sediments, species belonging to Methanoculleus, Methanogenium, and Methanolacinia have been isolated. From freshwater sediments, species belonging to Methanoculleus, Methanogenium, and Methanocorpusculum have been isolated. From rice roots and rice-field soils, species belonging to Methanoculleus have been isolated, and environmental clone sequences closely related to Methanoculleus and Methanogenium have been identified (Kudo et al. 1997). Methanomicrobium mobile has been isolated from bovine rumen (Paynter and Hungate 1968). Methanoplanus endosymbiosus lives as endosymbiont of the marine ciliate Metopus contortus (Bruggen et al. 1986).

3.4 Methanosarcinales

Members of the Methanosarcinales are widely distributed in marine and freshwater sediments, anaerobic digestors, and animal gastrointestinal tracts. Methanosarcina has been isolated from marine and freshwater sediments, anaerobic digestors, and rumen and has been detected in rice paddies (Chin et al. 2004; Krüger et al. 2005; Lu et al. 2005). Methanococcoides and Methanolobus have been isolated from aquatic environments with salinity near that of seawater. The habitats of Methanohalobium, Methanohalophilus, and Methanosalsum are restricted to hypersaline environments. Methanomethylovorans has been isolated from freshwater sediments and bioreactors. Methanosaeta has been isolated from freshwater sediments and anaerobic digestors and has been detected in rice paddies (Chin et al. 2004; Krüger et al. 2005) and marine sediments (Purdy et al. 2002). Methanimicrococcus has been isolated from cockroach hindgut and has been detected in anaerobic digestors (Weiss et al. 2008).

3.5 Methanocellales

All members of the Methanocellales have been isolated from rice soils, but they are also widely distributed in terrestrial ecosystems such as wetland soils and freshwater sediments based on environmental DNA sequence surveys (Conrad et al. 2006; Sakai et al. 2008, 2010; Lü and Lu 2012b). Methanocellales have been studied extensively in rice soils both in situ and in microcosms, revealing the following unique ecophysiological features. (i) They are closely associated with rice roots where they can actively convert plant-derived carbon into biomass and methane (Lu and Conrad 2005); (ii) they are able to tolerate the microaerophilic conditions around the rice roots, probably due to a robust antioxidant system encoded in their genomes (Erkel et al. 2006; Conrad et al. 2008; Sakai et al. 2011; Lü and Lu 2012a; Lyu and Lu 2017); (iii) they tend to become more active under low H2 but high temperature conditions (Lu et al. 2005; Wu et al. 2006; Peng et al. 2008; Sakai et al. 2009); and (iv) they frequently form syntrophic relationships with fatty acid degrading bacteria (Lueders et al. 2004; Liu et al. 2011; Rui et al. 2011; Gan et al. 2012). Additional ecophysiological features have also been revealed by studying Methanocellales in acidic peat soils, tank bromeliads and arid soils, suggesting that at least some members of Methanocellales could survive moderately acidic conditions, interact with plants other than rice such as Sphagnum in peat soil and tank bromeliads in neotropical forests, and tolerate desiccation (Sizova et al. 2003; Cadillo-Quiroz et al. 2010; Martinson et al. 2010; Angel et al. 2011, 2012).

3.6 Methanomassiliicoccales

Only one member of Methanomassiliicoccales has been isolated into pure culture from human feces (Dridi et al. 2012). Metagenomic analysis with human feces enrichment samples also revealed two new candidate species Candidatus ‘Methanomassiliicoccus intestinalis’ and Candidatus ‘Methanomethylophilus alvus’ (Borrel et al. 2012, 2013). This apparent common association with human suggests that Methanomassiliicoccales may play a role in human health. Due to their ability to metabolize trimethylamine into methane, it has been proposed that Methanomassiliicoccales may prevent or limit human diseases that are induced by trimethylamine (Brugere et al. 2014). However, distribution of Methanomassiliicoccales is not restricted to the human gut. An enrichment culture from anaerobic digester has led to the proposal of another candidate species Candidatus ‘Methanogranum caenicola’ (Iino et al. 2013). Environmental DNA sequence survey has suggested that Methanomassiliicoccales could be grouped into two clades, a gastro-intestinal tract clade that is largely associated with animal samples, and an environmental clade which includes mainly aquatic and terrestrial samples.

3.7 Other Methanogen Candidates

Methanogenesis pathways have been predicted from a euryarchaeon, Candidatus ‘Methanofastidiosa’, members of the newly proposed archaeal phyla Verstraetearchaeota and Bathyarchaeota (Evans et al. 2015; Nobu et al. 2016; Vanwonterghem et al. 2016). Candidatus ‘Methanofastidiosa’ belongs to the uncultivated WSA2 or Arc I cluster, which has long been identified as a core euryarchaeal group in anaerobic digestion that was previously thought to use H2/CO2 or formate for methanogenesis (Hendrickson et al. 2004; Nakamura et al. 2013). However, genomic data has now proposed that WSA2 methanogens may conduct methylated thiol reduction with H2 (Nobu et al. 2016). This suggests that they may be able to bridge the carbon and sulfur cycles, which may enable competition with CO2 reducing methanogens and sulfate reducers. Previously loosely classified as the Terrestrial Miscellaneous Crenarchaeota Group or TMCG, members of Verstraetearchaeota methanogens also had their first metagenomes reconstructed from anaerobic digesters, but environmental DNA sequence survey could extend their distribution to wetlands, freshwater sediments, and hydrocarbon-rich environments (Vanwonterghem et al. 2016). Previously known as the MCG or Miscellaneous Crenarchaeotal Group, the recently proposed Bathyarchaeota have been found in deep ocean and freshwater sediments, and they are particularly present in high abundance within sulfate-methane transition zones (Vetriani et al. 1999; Inagaki et al. 2003; Gagen et al. 2013; Evans et al. 2015). Likewise, their first metagenomes were recovered from coal-bed methane wells in an ocean basin (Evans et al. 2015). Although those novel methanogen candidates suggest the diversity of methanogens would be much higher than previously anticipated, interpretation of their environmental distribution and ecophysiology should be cautious. This is because no pure cultures have been available so far, and it remains elusive if every member of the WSA2, Verstraetearchaeota and Bathyarchaeota could also be capable of methanogenesis as predicted from a limited number of metagenomes.

4 Research Needs

A few established methanogen orders are still underrepresented by cultivated members. Methanocellales is only represented by one genus, and both Methanomassiliicoccales and Methanopyrales are represented by just one species. Discovery and isolation of new strains will certainly add to our knowledge of the diversity of those orders. Isolations of new strains are also necessary to support the classification of Methanimicrococcus blatticola and Methermicoccus shengliensis as separate families within the order Methanosarcinales and expand our knowledge of the diversity of Methanosarcinales. On the other hand, since the Methanosarcinales can use a relatively broad range of substrates for methanogenesis, isolation of new strains suitable for industrial purposes can be valuable.

Recent culture-independent studies have revealed the presence of novel phylogenetic groups of methanogens. Their isolation and characterization will also 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). In addition, many novel methanogen candidates are still only represented by metagenomes, such as the Candidatus ‘Methanofastidiosa’ and members of the archaeal phyla Verstraetearchaeota and Bathyarchaeota (Evans et al. 2015; Nobu et al. 2016; Vanwonterghem et al. 2016).

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. For instance, the genome-wide pairwise average nucleotide identity or ANI has been increasingly used to delineate species (Goris et al. 2007). However, convenient tools and methods will still need to be developed to meet the needs for analyzing large genome dataset. The Joint Genome Institute or JGI has been a pioneer in this filed, which has developed an Integrated Microbial Genome online pipeline to tackle the big data challenge (Markowitz et al. 2007a, b, 2009). Another grand challenge is to associate the environmental meta-data with the sequence data, which can provide enormous ecophysiological context for not only interpreting the sequence data from a single project but uncovering new trends across different projects.