1 Introduction

Rice is cultivated on approximately 155 million hectares worldwide, accounting for 14% of total arable land (Haefele et al. 2014). More than half of rice cultivation is under irrigated conditions. As a result, paddy fields are one of major anthropogenic sources for atmospheric methane (CH4), a potent greenhouse gas that has increased from about 715 ppb in the preindustrial times to 1850 ppb in 2015 (Saunois et al. 2016; Schaefer et al. 2016). The Intergovernmental Panel of Climate Change (2013) reported an annual emission of 33–40 Tg CH4 from the world paddy fields, equivalent to 12.5% of anthropogenic CH4, or 5.0% of annual global CH4 emission (IPCC 2013).

A campaign of CH4 flux measurements and observations in paddy fields initiated in the late 80s of last century. The consensus of numerous measurements from Europe, America, and across Asia revealed that the seasonal pattern of CH4 emissions from paddy fields comprises two or three peaks, with the first occurring in a few weeks after flooding and the second or third in the later season. The emission of CH4 is the result of three processes, namely, production, oxidation, and transportation. The production is processed by methanogens living in anoxic niches of paddy fields. Rice plants have the well-developed aerenchyma system that serves as the major conduit for the transportation of CH4 into the atmosphere. This aerenchyma system also allows the diffusion of O2 from the atmosphere to the roots and the surrounding soil, i.e., the rhizosphere. Thereby, the roots of rice plants and the rhizosphere become partially oxic and allow aerobic activity and especially the oxidation of CH4 that consumes on average a half of CH4 produced in the anoxic soils before emission (Conrad 2004; Liesack et al. 2000).

In correspondence to the seasonal pattern of CH4 emissions, the production of CH4 in paddy fields is considered comprising two phases. In the first phase, the soil organic matter, plant residues, and/or manures deposited from previous season provide the substrates for methanogenesis (Cicerone et al. 1992; Sass et al. 1991; Yagi and Minami 1990). In the later phase, the methanogenic substrates results mainly from the newly-generated plant materials, namely, the root exudates and sloughed-off root cells and debris (Holzapfelpschorn et al. 1986; Lindau et al. 1991). These two phases, often overlapping in reality, are assumed to correspond to the first and the second (and/or third) peak of CH4 emissions from paddy fields (Kimura 1997; Vandergon and Neue 1995). Obviously, the roots of rice plants play very important roles in CH4 emissions from paddy fields. In this chapter, I review the research advances of methanogenesis in association with rice roots with an emphasis on the finding and characterization of Methanocellales methanogens. Four aspects will be highlighted in particular: (1) the importance of root-derived C as a major C source for CH4 production in paddy fields; (2) identification of Methanocellales as the key methanogens responsible for CH4 production from the root-derived C; (3) phylogenetic and genomic characterization of the Methanocellales methanogens; and (4) mechanistic understanding of ecophysiology of Methanocellales in rice rhizosphere.

2 Importance of Root-Derived C in Methane Formation

Rhizosphere is the critical interface in terrestrial ecosystem. Through this interface, plants take up nutrients from soil and in return release photosynthesized products into the soil, feeding soil microbes. Microorganisms in the rhizosphere are actively involved in biogeochemical cycling of C, N, S, Fe, and many other elements (Arth et al. 1998; Lu et al. 2002; Neubauer et al. 2002; Scheid and Stubner 2001). It has been estimated that 30–60% of the net photosynthesized carbon is allocated to the roots, and 40–90% of this fraction is released into soil in the forms of root exudates, sloughed-off cells, and root debris or rhizodeposition to name together (Lynch and Whipps 1990). The rhizodeposition in paddy soils serves as a major carbon source for CH4 production. By using a 13C tracer approach, Minoda and Kimura (1994) revealed that part of photosynthesized 13C was transported to the rhizosphere, transformed to CH4, and emitted to the atmosphere just a few hours after the commencement of 13CO2 application (Minoda and Kimura 1994). Dannenberg and Conrad (1999) reported that about 3–6% of the assimilated radioactivity (14CO2) by rice plants were emitted as 14CH4 within 16 d after labeling (Dannenberg and Conrad 1999).

The rhizodeposition can be separated into different groups such as water-soluble exudates, secretions, lysates, mucilages, sloughed-off cells, decaying root debris, and gases (Bolton Jr. et al. 1993). It can contain all kinds of chemicals found in a plant cell, from sugars, amino acids, organic acids to more complex components such as proteins, polysaccharides, lipids, hormones, and vitamins. To evaluate the effect of rhizodeposition in methanogenesis, a comparative experiment was conducted using acetate and glucose as controls (Lu et al. 2000c). The effect of root exudates was found to be similar to acetate and glucose. But the addition of acetate and glucose yielded a significant priming effect on the decomposition of soil organic matter leading to a higher CH4 production, while root exudates caused only a moderate priming effect (Lu et al. 2000c).

To directly evaluate the effect of rhizodeposition on CH4 production and emission, the experiments under in situ conditions were conducted by observing the spatial variation of dissolved organic carbon (DOC) and dissolved CH4 in soil porewater along with a distance from rice roots (Lu et al. 2000a, b). These studies revealed that DOC in root zone soil, i.e., the rhizosphere, increased substantially with plant growth while that in the nonroot zone soil did not show significant change. Since no external organic materials were added, the increase of DOC in the root zone soil reflects the release of organic C from plant roots. The maximal concentration of DOC occurred between rice flowering and maturation, in consistence with the observation that root exudation of rice plants reached maxima during these stages (Lu et al. 1999). Dissolved organic C represents a mobile and labile form of soil organic matter and is expected to be easily degradable.

Correspondingly, the dissolved CH4 in the root zone soil began to increase at the maximum tillering stage of rice plants and reached to the maxima at the maturing stage (Lu et al. 2000a, b). In the nonroot zone, CH4 concentrations also increased gradually to the levels comparable with those in the root zone. But a lag period of 1–3 weeks was consistently detected. Higher dissolved CH4 in the root zone soil compared with nonroot zone soil (Fig. 1) suggests that CH4 in the root zone soil was produced locally from the decomposition of DOC pool derived from plant photosynthesized C. In correspondence with the concentration of dissolved CH4, the rate of CH4 emission increased significantly during the period from rice flowering to maturation. The statistical analyses revealed significant positive linear correlations between the porewater DOC, dissolved CH4, and the rate of CH4 emission over the growing season of rice plants (Lu et al. 2000a, b). These results support that the late season peaks of CH4 emission are due to the supply of plant-borne C through rhizodeposition (Neue et al. 1997).

Fig. 1
figure 1

Rice-plant microcosm for observing the dissolved organic C (DOC and dissolved CH4 in paddy soil. (a) Two sampling ceramic tubes are buried vertically beneath the soil surface with one close to root zone (i.e., rhizosphere) and another outside root zone; (b) Seasonal change of DOC in root zone (In) and outside root zone (Out); (c) Seasonal change of dissolved CH4 in root zone (In) and outside root zone (Out). (Taken from Lu et al. 2000b)

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Collectively, the pioneering studies demonstrated that the DOC pool in the rice rhizosphere was continuously enriched by plant-borne C during plant growth and this DOC pool is easily available for methanogenesis. The rice rhizosphere is probably a very important place for methanogenic activity. This finding however is apparently in conflict with the conventional theory that methanogens are known to be strictly anaerobic while the root surface and the closely connected rhizosphere is partly oxic due to O2 leaks from the plants. It remains elusive why certain methanogens can survive and even thrive in the rice rhizosphere.

3 Methanocellales as the Key Methanogens in Rice Rhizosphere

Two hypotheses were proposed to explain the activity of methanogenesis on rice roots and the rhizosphere: (i) methanogens colonizing rice roots are probably O2 resistant; (ii) they may develop a spatial strategy, inhabiting where O2 does not exist, for example, the old root segments where O2 release is lacking (Conrad 2004; Grosskopf et al. 1998b). Indeed, the community composition and activity of methanogenic archaea in association with rice roots differ greatly from those in the soil distant from roots. Specifically, the CO2 reduction pathway was found to be prevalent in CH4 production in rice root preparations (Chin et al. 2004; Conrad and Klose 1999, 2000; Lehmann-Richter et al. 1999), whereas the aceticlastic pathway usually accounted for over 65% of total CH4 production in the anoxic bulk soil (Conrad 1999; Conrad et al. 2002; Wind et al. 1999). Both environmental detection and enrichment cultivation from the excised rice roots revealed a dominance of an uncultured archaeal linage Rice Cluster I (Grosskopf et al. 1998b; Lehmann-Richter et al. 1999), which was later characterized as hydrogenotrophic methanogens and finally isolated into pure culture as a novel methanogen order Methanocellales (Lu and Lu 2012b; Sakai et al. 2008, 2010). The Methanosaeta spp. that often dominated in the bulk soil (Chin et al. 1999; Fey and Conrad 2003; Grosskopf et al. 1998a) was rarely detected on rice roots (Chin et al. 2004). These preliminary studies suggest that a very different population of methanogens are selected by rice roots. To identify the active methanogenic organisms responsible for CH4 production in rice rhizosphere, several experiments using molecular and isotopic labeling approaches were conducted (Lu and Conrad 2005; Lu et al. 2005).

3.1 Methanocellales on Rice Roots

In an incubation experiment using the excised rice roots as inoculants, the 13C fully labeled CO2 was applied with H2 or N2 in the headspace of incubation vessels (Lu et al. 2005). Two pH buffer systems based on carbonate or phosphate were prepared for incubation. The conditions thus created included the combination of buffer system phosphate (P) or carbonate (C) and the headspace composition H2 or N2. The 13CH4 was detected immediately after the anaerobic incubation of rice roots, indicating the readily activity of methanogens from rice roots. The production of CH4, however, was faster in C buffer than in P buffer. Strikingly, the rate of CH4 production was greater with N2 than with H2 in the headspace during the initiation period of methanogenesis. An estimate based on 13C labeling under C–N2 combination indicated that approximately 100% and 65% of CH4 were produced from CO2 reduction during the early and late periods, respectively. These estimates were consistent with previous reports showing the prevalence of hydrogenotrophic methanogenesis on rice roots (Conrad and Klose 1999; Conrad et al. 2002; Lehmann-Richter et al. 1999). The higher CH4 production under C–N2 compared to C–H2 combination indicates that the supply of H2 resulted in a negative effect on CO2-reducing methanogenesis in the incubations. This was somewhat surprising as H2 was the energy source for hydrogenotrophic methanogens to reduce CO2 for CH4 production.

The analysis of archaeal 16S rRNA gene abundances revealed a significant difference in community composition among different conditions. Under C–N2 condition, the Methanocellales, the yet-uncultured archaeal lineage by the time, showed a significant increase of 16S rRNA gene abundances in the 13C-labeled DNA, indicating that these methanogens were more active than others. Apparently, the Methanocellales were responsible for CH4 production from H2/CO2, the dominant pathway of CH4 production in rice root preparations. The relative abundance of Methanosarcinaceae also increased in the late stage, indicating the increasing contribution of acetate-dependent methanogenesis towards the end of incubation (Chin et al. 2004; Conrad et al. 2002). When H2 was supplied (i.e., under C–H2), the Methanosarcinaceae became exclusively dominated, whereas Methanocellales were detected only at low abundance. The high H2 condition apparently favored the growth of hydrogenotrophic Methanosarcina spp. over Methanocellales. Under P buffer conditions, the Methanobacteriaceae and Methanosarcinaceae were selected, while Methanocellales were present only marginally.

The Methanocellales had been repeatedly detected in different environments including rice roots, anoxic rice soils (Chin et al. 2004; Grosskopf et al. 1998a; b; Lueders and Friedrich 2000), and wetlands (Galand et al. 2002; Jurgens et al. 2000; Sizova et al. 2003). Little had been known however about their physiology. The above DNA-SIP experiment revealed that these methanogens were remarkably suppressed when H2 was supplied to either P or C buffer systems. A previous enrichment study showed that phosphate was not toxic to Methanocellales (Lehmann-Richter et al. 1999). Therefore, application of H2 appeared the only reason for the depression of Methanocellales in root preparations. This finding increased the clouding in understanding methanogenesis associated with rice roots. It was speculated that Methanocellales were probably adapted to low H2 condition and were less selective under the artificially H2-enriched conditions. It has been reported that the H2 partial pressure can indeed regulate the expression of genes involved in methanogenesis (Luo et al. 2002) that can vary depending on methanogen identity.

3.2 Methanocellales in Rice Rhizosphere

The unculturability of vast microbial species in environments demands culture-independent approaches to understand their activity and functioning. The development of stable isotope probing (SIP) in combination with molecular fingerprinting based on DNA and RNA provided such a powerful approach (Lu and Conrad 2005). This technique has been used to detect methanogens in rice root preparations as described above (Lu et al. 2005). To identify the active methanogens in rice rhizosphere under in situ conditions, RNA-SIP approach was applied to in an intact rice-soil system, in which rice plants were supplied with the 13C-labeled CO2 for plant photosynthesis and the photosynthesized 13C was tracked for its distribution from the plant top to the rhizosphere and the assimilation by soils microbes.

In this plant-soil microcosm, CH4 in soil pore water as well as that emitted into the air was found to be rapidly labeled with 13C (Lu and Conrad 2005), suggesting that methanogenesis in the rice rhizosphere was active and closely linked to plant photosynthesis under in situ conditions. The 13C labeled RNA retrieved from rice rhizosphere revealed a signature fingerprint associated with methanogenic archaea (Fig. 2). Specifically, a characteristic terminal restriction fragment (394-bp) was significantly enriched with 13C out of seven fragments belonging to different archaeal lineages (Lu and Conrad 2005). By comparison, no specific signature fingerprint was revealed in the control microcosm without 13CO2. Undoubtedly, the methanogenic archaeal lineage characterized by the signature fragment 394 bp assimilated the 13C derived from organic substances that were deposited into the rhizosphere after photosynthesis. To characterize phylogenetic affiliation of this active methanogen in rice rhizosphere, the 16S RNA clone libraries were constructed, which revealed that out of seven methanogen lineages, the Methanocellales methanogens (i.e., uncultured RC-I by that time) was characterized with the signature fragment 394-bp (Lu and Conrad 2005). Thus, the Methanocellales were identified as the most active methanogens in rice rhizosphere where the release of organic substrates and O2 leaks occur simultaneously. These results are in line with earlier studies showing that CH4 production in excised rice root preparations is mainly due to the activity of Methanocellales (Lehmann-Richter et al. 1999; Lu et al. 2005; Lueders et al. 2001). Given the fact that paddy fields are an important source of methane emission (Conrad 2009; IPCC 2013) and plant-photosynthesized carbon provides a major source for CH4 production in paddy soil (Lu et al. 2000a, b; Minoda and Kimura 1994), the identification of Methanocellales as the key player in rice rhizosphere opens a new window for further investigation and deeper understanding of methanogenesis in paddy fields.

Fig. 2
figure 2

Rice-plant microcosm for RNA-SIP detection of active methanogens in rice rhizosphere. (a) Rice plants were fed with 13CO2 in a closed chamber and microbial RNA were extracted from rice rhizosphere for RNA-SIP dissection; (b) Fingerprinting of the density resolved RNA revealed that a signature fragment (394 bp), representative of Methanocellales (Rice Cluster I), was 13C labeled. (Taken from Lu and Conrad 2005)

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4 Metagenomic Insights into Methanocellales’ Adaptation to Rice Rhizosphere

After the discovery of Methanocellales as the key player of CH4 production in paddy soils, it was highly demanding to elucidate the physiological mechanisms of their activity, particularly in a way associated with rice roots. Due to the nature of difficulty in isolating them into pure cultures, enrichment cultivations were intensively tried in the Max-Planck Institute for Terrestrial Microbiology that finally resulted in an enrichment, named MRE50, in which Methanocellales were the only archaeal component (Erkel et al. 2005). This enrichment was then served as a genomic source for constructing fosmid clone library in order to pinpoint the Methanocellales metagenome (Erkel et al. 2006). A complete genome sequence of a single Methanocellales representative (RC-IMRE50) was reconstructed that offers the path to look into the putative metabolic capacity of Methanocellales methanogens.

The RC-IMRE50 genome has a size of about 3.18 Mb with 3103 predicted coding sequences. The genome reveals a series of unique features for energy conservation, biosynthesis, C and N metabolisms that are distinct from many known methanogens (Erkel et al. 2006). The central energy metabolism with CH4 production from CO2 reduction appears related to the hydrogenotrophic Methanosarcina, containing a membrane-bound hydrogenase with cytochrome b, a trait found only in the members of Methanosarcinales by the time (Thauer 1998). However, unlike Methanosarcina spp., RC-IMRE50 also encodes a system of using formate and formaldehyde for methanogenic growth, which is the typical trait of obligately hydrogenotrophic methanogens (Erkel et al. 2006). RC-IMRE50 harbors adenosine 5′-monophosphate-forming acetyl-coenzyme A (CoA) synthetase (ACS) for acetate assimilation and the carbon monoxide dehydrogenase complex for acetyl-CoA biosynthesis from CO2 that are common to most obligately hydrogenotrophic methanogens. But RC-IMRE50 additionally encodes a membrane-bound pyrophosphatase that can help these methanogens to recover a portion of the energy invested in acetate activation, which is not available in other methanogens that use ACS for acetate assimilation.

The pyruvate metabolism encoded in RC-IMRE50 includes ethanol production from acetaldehyde, acetoin production from acetolactate, and two pathways for acetyl-CoA formation from pyruvate. Most anaerobes including methanogens use the pyruvate-ferredoxin oxidoreductase that is oxygen-sensitive for the decarboxylation of pyruvate and acetyl-CoA production. By comparison, aerobes usually use the pyruvate dehydrogenase (PDH) for similar function. Interestingly, the RC-IMRE50 genome encodes both pathways. The PDH complex has been typically found in aerobic and facultatively anaerobic microorganisms but is lacking in all known methanogens by the time. It was therefore speculated that RC-IMRE50 likely uses the glycolytic pathway to survive the oxic periods (Erkel et al. 2006). Energy for maintenance may result from pyruvate and acetate production. Reducing equivalents generated from glucose and pyruvate oxidation can be recycled through the fermentation of pyruvate to ethanol. The allosteric control of the glycolytic pathway may allow RC-IMRE50 to respond quickly to the environmental changes in redox states.

The RC-IMRE50 genome appeared to contain biosynthetic pathways for all amino acids except glutamate (Erkel et al. 2006). But the glutamate synthesis was later found to be present in the genome analysis of Methanocella pure cultures (see details below). Nevertheless, RC-IMRE50 encodes a candidate ABC-type glutamate import system. The ability of RC-IMRE50 to take up glutamate from environments and to incorporate it into enzyme synthesis was experimentally confirmed (Erkel et al. 2006). This feature might confer an advantage for Methanocellales to live near rice roots as glutamate may be available in root exudates and/or decomposing plant root materials. Besides the glutamate uptake, RC-IMRE50 genome reveals two additional mechanisms for nitrogen acquisition via ammonium assimilation and dinitrogen fixation (nitrogenase). These combined traits indicate the metabolic flexibility of RC-IMRE50 in nitrogen acquisition. In addition, RC-IMRE50 also reveals an unique sulfur assimilation through the reduction of sulfate to sulfide. It contains genes coding for sulfurylase and adenylylsulfate kinase that are lacking in all methanogen genomes sequenced by the time. Most methanogens depend on sulfite, sulfide, or sulfur-containing amino acids as sulfur source for assimilation. The ability of RC-IMRE50 to use sulfate may confer Methanocellales another advantage to adapt the rhizospheric environment, where sulfate instead of the reduced sulfur forms may be available due to oxic conditions.

Since O2 is diffused from the top of rice plants down to roots and released into the rhizosphere, the transient anoxic/oxic conditions prevail on root surface and in the rhizosphere soil. In addition, paddy fields often experience wet-dry cycling due to field management requirement (Liu et al. 2015). The key for methanogens to inhabit rice rhizosphere is therefore dependent on the capacity of resisting oxidative stresses. Aerotolerant systems were previously found in the aceticlastic Methanosarcina spp. The obligately hydrogenotrophic methanogens however acquire only a limited set of antioxidant enzymes. Strikingly, the RC-IMRE50 genome encodes multiple sets of genes coding for antioxidant enzymes, including the mono-functional large subunit heme catalase that is most ancient and robust of all known catalases (Chelikani et al. 2004). Three different reactive oxygen species (ROS) scavengers are present that can be used to remove both external and internal superoxide anions. In particular, the exogenous superoxide anions can be scavenged by a periplasmic Cu, Zn-dependent superoxide dismutase (SodC) (Fournier et al. 2003), while the cytoplasmic superoxide anions be removed by two types of superoxide reductase (SOR) containing rubredoxin and desulfoferrodoxin, respectively. SORs are considered the most important oxygen defense systems in anaerobes (Jenney et al. 1999), especially under strong oxygen exposure (Fournier et al. 2003). In addition, the RC-IMRE50 also encodes bacterial-type enzymatic systems with repair mechanisms for oxidative lesions of DNA, such as formamidopyrimidine-DNA glycosylase (MutM), 3-methyladenine-DNA glycosylase (MPG), and the Holliday junction resolvasome (RuvABC) (Erkel et al. 2006). Possessing these multiple antioxidant and repair systems confers Methanocellales the extraordinary ability to be aerotolerant. Thus, Methanocellales are genetically equipped with competitive advantages over obligately hydrogenotrophic methanogens in the rice rhizosphere. Together with the potentials of acquiring alternate sulfur and nitrogen nutritions, Methanocellales appear to have evolved the methanogenic life well-fitting to the rice rhizosphere.

5 Isolation of Methanocella Species into Pure Culture

Despite the metagenomic insights into their adaptation to rice rhizosphere and more generally to oxic conditions, deeper understanding of their physiology and ecology is impossible without isolation of Methanocellales into pure culture. The efforts to isolate them therefore have never been stopped though the difficulty. The first pure culture of Methanocella were obtained from a Japanese rice field soil using a syntrophic cultivation approach. The formal order name, Methanocellales, was then given based on the phylogeny of this pure culture, and the strain itself was named as Methanocella paludicola strain SANAET (Sakai et al. 2007, 2008). The second isolate, a thermophilic methanogen, Methanocella arvoryzae strain MRE50T, was later purified from the enrichment established for the metagenomic investigation (Lueders et al. 2001; Sakai et al. 2010). The isolation of these two strains would have offered a chance to address many ecophysiology questions. Unfortunately, despite the successful isolation of strains SANAET and MRE50T, the maintenance and cultivation of these strains in lab require some extraordinary techniques, which impede the further investigations. Therefore, more isolates particularly with the fast-growing trait are still needed. Such a strain, Methanocella conradii strain HZ254T, named after Ralf Conrad, a pioneering scientist on this methanogen lineage, was finally obtained from a Chinese paddy field soil (Lu and Lu 2012b). A moderate high temperature has been an effective strategy to isolate this strain, in line with early enrichment studies (Fey et al. 2001; Peng et al. 2008).

The third strain was phylogenetically closer to M. paludicola SANAET (16S rRNA gene similarity of 95.0% and mcrA gene similarity of 87.5%) than to M. arvoryzae MRE50T (92.4% and 86.5% for the 16S rRNA and mcrA similarity, respectively) (Fig. 3) (Lu and Lu 2012b). Though three strains share some common phenotypic features, such as the rod-shaped morphology, they differ in formate utilization, flagellum formation, temperature optimum, pH range, and salinity susceptibility. In contrast to the phylogenetic relationship, strain HZ254T seems to be closer to MRE50T than SANAET in major phenotypic traits including temperature optimum, flagellum formation, and salinity susceptibility. The 16S rRNA gene sequence divergence of 5% between HZ254T and SANAET implies that strain HZ254T potentially represents a new genus instead of new species (Lu and Lu 2012b).

Fig. 3
figure 3

Phylogeny of Methanocellales based on (a) 16 S rRNA genes and (b) Deduced methyl-coenzyme reductase subunit A amino acid sequence (McrA). (Taken from Lu and Lu 2012b)

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6 Comparative Genomics and Comprehensive Understanding of Methanocellales

6.1 Phylogeny and Taxonomy

Thus far three Methanocella strains have been available, namely M. paludicola SANAET, M. arvoryzae MRE50T and M. conradii HZ254T (Lu and Lu 2012b; Sakai et al. 2008, 2010). Though they have been classified together as a genus Methanocella, they could potentially represent multiple genera and even families due to low similarities of their 16S rRNA and mcrA genes (Lu and Lu 2012b; Sakai et al. 2010). In consistence with the analyses of 16S rRNA and mcrA, the global nucleotide identities calculated based on whole genome alignments suggest that M. conradii and M. paludicola are more closely related each other than to M. arvoryzae. The phylogenetic trees constructed based on multiple markers (i.e., 16S rRNA, mcrA and ribosomal proteins) also placed M. conradii closer to M. paludicola than to M. arvoryzae (Borrel et al. 2013; Lu and Lu 2012b). The Average Amino Identity (AAI) that can be more relevant to infer genetic relationship at high taxonomic levels indicated that M. conradii and M. paludicola together represent a genus, while M. arvoryzae alone represents a separate genus (Lyu and Lu 2015), according to the consensus criterion of AAI (Konstantinidis and Tiedje 2007).

Before the proposal of order Methanocellales, methanogens had been classified into five characterized orders, i.e., Methanopyrales, Methanococcales, Methanobacteriales, Methanomicrobiales, and Methanosarcinales (Liu and Whitman 2008). Comparative genomic analyses have grouped these orders into Class I (consisting of Methanopyrales, Methanococcales and Methanobacteriales), Class II (the Methanomicrobiales), and Class III (the Methanosarcinales) methanogens, respectively (Anderson et al. 2009). Phylogenetically, the Methanocellales can be placed between Class II and III methanogens (Lu and Lu 2012b; Sakai et al. 2008, 2010). Although the physiological relationships remain unclear, Methanocellales do share some ecological features with either Class II or III. For instances, both Methanocellales and Class II methanogens are detected in rice soils and wetlands where H2 partial pressure is low (1–10 pa), whereas Methanocellales also share common habitats with Class III methanogens, such as upland soils where aeration and desiccation occur periodically (Angel et al. 2012; Angel et al. 2011; Aschenbach et al. 2013; Conrad et al. 2006).

Genome sequences of three Methanocella strains and their comparative analysis offer an opportunity to elucidate the basic ecophysiology traits of this novel type of methanogens. A detailed reannotation of SANAET and MRE50T was performed using the same annotation pipeline used for the third strain HZ254T to ensure the consistency in comparison (Lyu and Lu 2015). The reannotation of SANAET and MRE50T genomes revealed several new genes, pseudogenes, and some CRISPR region(s) that were not identified previously. Analyses of COG, Pfam, and TIGRfam classifications also revealed more functional insights into many genes not assigned before. Whole genome alignments revealed the extensive rearrangements of genomic regions among three strains. Three Methanocella strains share a core genome comprised of 1187–1245 ortholog groups, depending on the threshold set for amino acid identity (Lyu and Lu 2015). More orthologs are shared between M. conradii and M. paludicola than to M. arvoryzae, consistent with the phylogenetic relationship among them.

6.2 Novel Features of Core Metabolisms for Methanogenesis

All three strains possess a complete gene set for the typical hydrogenotrophic methanogenesis characterized as the closed Wolfe cycle (Thauer 2012). The major differences among three genomes are the copy numbers of several genes in the pathway, specifically the genes coding for the B subunit of F420-reducing hydrogenase (frhB), the D subunit of F420-nonreducing hydrogenase (mvhD), and the E subunit of energy-converting hydrogenase (echE). The ecological insights into these differences have yet to be evaluated. Two novel features, however, were identified that are shared by all three strains (Lyu and Lu 2015). The first is the gene organization related to the Wolfe cycle and the second is the presence of a putative [NiFe] hydrogenase complex that was not found in other methanogens.

Hydrogenotrophic methanogens are known to employ a multienzyme complex to perform the flavin-based electron bifurcation for the energy conservation from oxidation of H2 or formate (Costa et al. 2010; Lie et al. 2012). This complex consists of formylmethanofuran dehydrogenase (Fwd), heterodisulfide reductase (Hdr), and Mvh (Fwd/Mvh/Hdr in short). The formate dehydrogenase (Fdh) may also join with the formation of the Fwd/Mvh/Fdh/Hdr supercomplex. In Class I methanogens, though the formation and functioning of Fwd/Mvh(Fdh)/Hdr multienzyme complex, the genes coding for these components are located separately in their genomes (Hendrickson et al. 2004; Kaster et al. 2011; Thauer et al. 2010). In contrast, Methanocella as well as many of Class II methanogens organize most of those genes into large gene clusters. A 10-gene cluster consisting of whole sets of fwd and hdr genes and a gene for the subunit D of Mvh (mvhD) was identified in all three Methanocella stains (Mtc_2477–2468, MCPlv_2811–2802, and MRE50lv_2189–2180) (Lyu and Lu 2015). There exists even a second larger gene cluster comprising the above 10 genes together with two fdh genes in M. arvoryzae and M. paludicola (MCPlv_1593–1604 and MRE50lv_0274–0285). This unique organization of large gene cluster may facilitate the assembly of multienzyme complex with less biological cost and preventing the transcriptional resource waste (Anderson et al. 2009; Lie et al. 2012). The inclusion of fdh in the gene cluster may allow M. arvoryzae and M. paludicola to grow on formate as the sole carbon and energy source, whereas M. conradii is not known to have this ability (Lu and Lu 2012b).

Methanocella seem to be exceptionally adapted at low H2 concentrations. This feature is initially illustrated in root preparation experiment (Lu et al. 2005). The isolation of the strain M. paludicola SANAET by using the syntrophic coculture technique confirmed that low H2 condition favors Methanocella over other hydrogenotrophic methanogens (Sakai et al. 2007). More evidences are illustrated with the detection of Methanocellales in association with different bacteria syntrophs that syntrophically oxidize short-chain fatty acids in paddy soils (Gan et al. 2012; Liu et al. 2011; Lueders et al. 2004; Rui et al. 2011). Therefore, though the Class I methanogens use the similar Wolfe cycle and perform the flavin-based electron bifurcation for the core metabolisms, Methanocellales appear to possess a specific capacity to perform these functions at H2 level close to the thermodynamic limit. The reason for this unique feature is possibly related to the presence of the large gene cluster coding for Fwd/Mvh/Hdr complex, which can confer a better efficiency in energy conservation through facilitating the assembly of multienzyme complex for electron bifurcation. Gene clustering is considered a common strategy used by prokaryotes to increase efficiency in forming protein complexes (Sneppen et al. 2010). A global transcriptional analysis for HZ254T indeed illustrated the elevated expression of this gene cluster under limited H2 condition in syntrophic coculture compared with high H2 in monoculture (Liu et al. 2014).

The second unique feature of Methanocella genomes is the presence of a putative [NiFe] hydrogenase complex. The coding genes for this complex are organized into a 8-gene cluster (Mtc_0479–0486, MRE50lv_2279–2272 and MCPlv_2682–2674) including echE (energy-converting hydrogenase subunit E) and hdrB homologs (heterodisulfide reductase subunit B) (Lyu and Lu 2015). The EchE homologs possess the [NiFe] binding motifs and are phylogenetically more closely related to the bacterial Coo hydrogenase (carbon monoxide-induced hydrogenase) in the sulfate-reducing bacteria than to the canonical Ech hydrogenase in methanogens. A significant divergence from Coo and Ech is that the novel hydrogenase does not encode the Na+/H+ translocating subunit (i.e., CooM or EchA), while all other subunits essential for the oxidation of H2 and electron transfer are present (Lyu and Lu 2015). Similar to the phylogeny of Ech, the HdrB homologs are phylogenetically more closely related to homologs in sulfate-reducing prokaryotes than to those in methanogens. Compared to the canonical form that catalyzes CoB-S-S-CoM reduction in methanogens, HdrB homologs in sulfate-reducing prokaryotes are involved in sulfite reduction and presumably reduce the intramolecular disulfide bridge of the DsrC (Dissimilatory sulfite reductase subunit C) (Grein et al. 2013). Based on phylogeny and traits described above, the novel hydrogenase is tentatively named as the Disulfide Reducing Hydrogenase (Drh) complex (Lyu and Lu 2015). Due to the absence of the Na+/H+ translocating subunit, Drh would be unable to conserve energy from H2 oxidation. It was speculated that the HdrB subunit in the Drh complex may use the disulfide of an unknown enzyme or compound as the electron acceptor (Aslund et al. 1997).

Methanocellales appears to have exceptional aerotolerant abilities, and all three strains encode a substantial number of genes involved in antioxidant resistance (Erkel et al. 2006) (and see below for further information). However, a robust antioxidant system would need to consume a number of reducing equivalents (Imlay 2008). The Wolfe cycle is unlikely to provide such a source, because its activity shall be severely repressed under oxic conditions. Given the close phylogenetic relationship of Drh to Coo and Fhl (formate-hydrogen lyase) that are known to be involved in CO detoxification and stress resistance (Bonam et al. 1989; Rossmann et al. 1991), Drh in Methanocella is probably involved in the antioxidant tolerance. It has been revealed that methanogens tend to develop their antioxidant systems around thioredoxins using the thio/disulfide redox cycling mechanism (Susanti et al. 2014). The oxidation of thio mosaics into disulfide in cells would be expected under air exposure. Methanocella perhaps use Drh to couple the H2 oxidation (i.e., electron supply) to thio/disulfide redox cycling (i.e., via the HdrB) and channel the electrons into repairing machinery for oxidation damages.

6.3 Carbon Metabolisms

All genes for the Embden-Meyerhof-Parnas (EMP) pathway except hexokinase or glucokinase are present in three Methanocella strains, indicating that they are able to convert glucose-1-phosphate into pyruvate via glycolysis. The presence of ppsA (phosphoenolpyruvate synthase) and suhB (D-fructose 1,6-bisphosphatase) indicates that they also have the ability of synthesizing glucose-1-phosphate from pyruvate through gluconeogenesis that may further lead to the synthesis of glycogen, a reserve material in many methanogens (Yu et al. 1994). Thus, under certain circumstances Methanocellales may use gluconeogenesis to store energy and switch to glycolysis under starvation.

Pyruvate plays a pivotal role in cellular chemistry. Methanocella appear to have diverse pathways for pyruvate metabolisms. Firstly, all three strains could reversibly oxidize pyruvate to acetyl-CoA using pyruvate ferredoxin oxidoreductase (Por) and/or pyruvate dehydrogenase (Pdh). Acetyl-CoA can then be converted to acetate by acetyl-CoA synthase (Acd) or vice versa by acetyl-CoA synthetase (Acs). The presence of Ppa (inorganic pyrophosphatase) would allow Methanocella to recover a portion of energy via proton translocation during the acetate activation for biosynthesis. Though physiological tests indicate that acetate is needed for growth by all three strains (Lu and Lu 2012b; Sakai et al. 2008, 2010), M. arvoryzae may use the Codh/Acd (CO dehydrogenase/acetyl-CoA synthase) for autotrophy. As indicated earlier, Pdh is known to operate mainly in aerobic and facultatively anaerobic microorganisms while Por is oxygen sensitive. Comparative genomic analysis confirms that Pdh is present in all three strains (Lyu and Lu 2015). Possessing of both For and Pdh by Methanocella possibly offers them an adaptive strategy to the alternating anoxic/oxic conditions. Specifically, Pdh is probably activated for pyruvate metabolism under oxic conditions (Erkel et al. 2006; Sakai et al. 2011). Secondly, all three strains possess the coding genes for acetolactate synthase, which could be used in biosynthesis of branched-chain amino acids from pyruvate (Bowen et al. 1997). A third potential pathway of pyruvate metabolism probably uses Pdc (pyruvate decarboxylase) to ferment pyruvate into either ethanol to recycle NAD or into acetate to generate reduced ferredoxin, which however was detected only in M. arvoryzae and the annotation for the coding genes was putative due to the low identity to known pdc. Further experimental studies are necessary to verify different pathways of pyruvate metabolisms in Methanocella.

Initial metagenomic and genomic surveys indicated that only the coding genes for isocitrate dehydrogenase and fumarase were present in Methanocella, leading to the assumption that neither the oxidative nor the reductive tricarboxylic acid (TCA) cycle was operated in Methanocellales (Erkel et al. 2006; Lu and Lu 2012a; Sakai et al. 2011). Due to the possible lacking of 2-oxoglutarate (2-OG) that is needed in glutamate synthesis, Methanocella may need to acquire glutamate from environments. A careful manual annotation of three Methanocella genomes, however, revealed that all three strains possess the (Re)-type citrate synthase homologs (Mtc_1389, MRE50lv_1257, and MCPlv_0455), sharing an identity of ~33% to that of Clostridium kluyveri (Lyu and Lu 2015). The manual annotation also identified a putative aconitase in all three strains encoded by two genes belonging to COG1679 and COG1786. These two genes located in a same cluster would presumably produce the functional motifs in one type of aconitate hydratase, aconitase A. Two types of aconitate hydratase are known: aconitase A widespread in all three domains of life while aconitase B found only in Proteobacteria (Makarova and Koonin 2003). Collectively, the manual reannotation suggests that Methanocella encode the nonconventional citrate synthase and aconitate hydratase, and together with the isocitrate dehydrogenase, a partial oxidative TCA from citrate to 2-oxoglutarate (2-OG) would be possible for Methanocella (Lyu and Lu 2015).

6.4 Nitrogen Metabolisms

Methanocella encode diverse nitrogen assimilation and regulation systems with a few differences among three strains (Lyu and Lu 2015). They all encode Amt (ammonia transporter) for ammonia uptake, which can then be assimilated via the GS (glutamine synthetase) and GOGAT (glutamate synthase) systems. GDH (glutamate dehydrogenase) that usually operates at high ammonium concentration is also present in M. arvoryzae and M. paludicola, increasing their flexibility for ammonium assimilation. At least one amino acid ABC transporter is identified in each strain, allowing them to uptake organic nitrogen sources. A complete nif operon for nitrogen fixation is present in M. conradii and M. arvoryzae, but not in M. paludicola (Lyu and Lu 2015). Thus, nitrogen fixation may operate in some but not all Methanocellales methanogens.

A 2-OG (2-oxoglutarate) based nitrogen regulation system is predicted in three strains (Lyu and Lu 2015). This system senses nitrogen level using 2-OG as a trigger as having been revealed in Methanococcus and Methanosarcina (Leigh and Dodsworth 2007). When nitrogen is limiting, 2-OG accumulates that removes the inhibitory effects of GlnK (nitrogen regulatory protein P-II) on Amt and GS and of NifI1I2 on Nif (nitrogenase), hence promoting both ammonium uptake and N2 fixation. In addition, the enhancement of GS activity by 2-OG accelerates nitrogen assimilation. In addition, NrpR is also found in three strains. NrpR is a transcription repressor that is found mainly in Archaea (Lie et al. 2007; Lie and Leigh 2007). In nitrogen-starved cells, 2-OG would prevent NrpR from binding to the operators in the promoter regions of nif and glnA, hence facilitating transcription of these nitrogen assimilation genes. Though the identification of the 2-OG based nitrogen regulatory system, whether it functions and plays a role in N nutrition has yet to be determined by experimental studies.

6.5 Sulfur Metabolisms

The metagenomic analysis of RC-IMRE50 has revealed the presence of a complete set of genes for sulfate assimilation, namely, the cysC (adenylylsulfate kinase), cysH (PAPS reductase), and sulfite reductase (Erkel et al. 2006). This prediction is reconfirmed in the genomes of M. arvoryzae and M. paludicola (Sakai et al. 2011). But the gene coding for sulfite reductase is missing in M. conradii (Lu and Lu 2012b). Nevertheless, all three strains encode a PiT family transporter for the uptake of phosphate or sulfate, and M. arvoryzae additionally encodes a putative sulfate permease. Thus, at least M. arvoryzae and M. paludicola are likely able to use sulfate as a sulfur source. For FeS cluster assembly, sulfite is often the only sulfur source for many methanogens due to the lack of cysteine desulfurase, whereas the genes coding for this enzyme are present in Methanocella (Lyu and Lu 2015). In addition, three strains encode two iron sulfur assembly systems which enable them to explore alternative sulfur sources for FeS synthesis. The first uses ApbC type FeS carrier and SufBCD type synthesis system, which is present predominantly in Class I methanogens with sulfide as sulfur source, while the second uses the A-type FeS carrier and IscSU synthesis system with cysteine as sulfur source (Liu et al. 2012). This may allow Methanocella to switch between two systems in concert with redox changes in environment, using sulfide at low and cysteine at high redox potentials, securing sulfur nutrition. The putative use of sulfate and the presence of two iron sulfur assembly systems reinforce the adaptation of Methanocellales to oxidative conditions.

6.6 Understanding of Oxidative Adaptation

Methanogenic analysis has indicated that Methanocellales contain multiple sets of genes coding for antioxidant systems that is the key for surviving and thriving in alternate anoxic/oxic habitats like rice rhizosphere. To confirm this capacity, an extensive comparative genomic analysis was conducted for three strains (Lyu and Lu 2018). Theoretically, three lines of antioxidant strategies could have been evolved in microbes to defend the oxygenation challenge: (i) avoiding the production of reactive oxygen species (ROS), (ii) reducing accumulation of ROS within the cell, and (iii) repairing self for ROS damage. Studies have revealed that these strategies are essential for both aerobes and anaerobes to survive oxidative stress (Imlay 2008, 2015). The comparative genomic analysis therefore has been focused on identifying these strategies in Methanocella genomes (Lyu and Lu 2018).

The methanogenesis pathway where redox reactions are most active inside the cell of methanogens is assumably the main place for ROS production. Specifically, the flavin-based electron bifurcation system that requires the formation of flavosemiquinone could react with oxygen to form O2− and H2O2 (Buckel and Thauer 2013). This electron bifurcation mechanism has been proposed to operate in Methanocella (Liu et al. 2014; Liu and Lu 2018). The comparative genomic analyses indicate that the number of [4Fe-4S] motifs involved in the electron bifurcation-based methanogenesis was reduced by about 70% in Methanocella compared to the Class I methanogens (Lyu and Lu 2018). This change in electron transfer machinery could reduce the chance for HO· production through the Fenton reaction.

The second major strategy lies on the capacity of O2/ROS elimination that is catalyzed by a variety of antioxidant enzymes in microbes. The enzymes known to reduce O2 to H2O and transform H2O2 and O2− to less toxic O2 have been characterized (Imlay 2008). Many of O2/ROS eliminations depend on redox reactions and require reducing power to proceed. Small redox proteins play an important role in supplying such a reducing power (Lu and Holmgren 2014). These proteins also serve as a buffering system to keep cellular redox system from becoming over oxidized (Susanti et al. 2014). Though the presence of O2/ROS elimination enzymes in many methanogens, Methanocella possess statistically more genes encoding these enzymes than the Class I (hydrogenotrophic) methanogen counterparts (Lyu and Lu 2018). These observations suggest that Methanocella are equipped with a higher capacity for O2/ROS elimination (Fig. 4).

Fig. 4
figure 4

Methanocellales contain on average highest numbers of genes encoding antioxidant systems. Included for the comparative analysis are three genomes of Methanocellales [Mc(3)], nine genomes of Methanosarcinales [Ms(9)], seven genomes of Methanomicrobiales [Mm(7)], eight genomes of Methanobacteriales [Mb(8)], thirteen genomes of Methanococcales [Mcc(13+)] and one genome of Methanopyrales [Mp(1)]. The coding genes for analysis consist of catalase (kat), superoxide dismutase (sod), peroxiredoxin (prx), superoxide reductase (sor), F420H2 oxidase (fpr), thioredoxin (trx), glutaredoxin system (glx) and rubredoxin (rbx). The number in parentheses indicates the COGs of the respective genes

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A closer examination of the O2/ROS elimination systems indicates the evolutionary robustness of this elevated capacity in Methanocellales. First, NO/O2 reductase is more abundant than F420H2 oxidase in Methanocella. Both enzymes can oxidize O2 into H2O, but the latter is deactivated when cells are exposed to air (Seedorf et al. 2004), while the former has a higher Km for O2 (Silaghi-Dumitrescu et al. 2005). In addition, NO/O2 reductase detoxifies NO, a product of denitrification that can be produced at the oxic-anoxic interface (Kluber and Conrad 1998). A shift from F420H2 oxidase to NO/O2 reductase could suggest an evolutional adaptation of Methanocellales to the severer oxidative conditions. Second, rubredoxin and thioredoxin are the major small redox proteins found in methanogens. Thioredoxin operates at much lower redox potentials than rubredoxin, transferring electrons at around −300 to −120 mV versus 0 ± 100 mV, respectively (Aslund et al. 1997; Lin et al. 2005). In comparison with other hydrogenotrophic methanogens, Methanocella contain similar number of rubredoxin proteins, but the thioredoxin proteins are substantially increased (Lyu and Lu 2018), indicating a potential enhancement of the redox buffering system in Methanocellales. Third, transmembrane thioredoxin proteins are present in Methanocella, but rare in other hydrogenotrophic methanogens (Table 2). In addition to the presence of thioredoxin domain both in the cytoplasmic and periplasmic side, these transmembrane proteins have two or three cysteine residues in the transmembrane region. These transmembrane thioredoxin proteins may enable electron shuffle between the cytoplasmic and periplasmic spaces, which may help with redox recovery around the cellular membranes under oxidative stress. Fourth, while the classical hydrogenotrophic methanogens appear to use F420H2 to regenerate the reduced thioredoxin, Methanocellales probably use NADPH or NADH. NADPH or NADH are more stable electron carriers than F420H2 in an oxygenated Earth environments. These changes in oxidant-detoxifying systems of Methanocellales appear systematic and holistic.

The third strategy for oxidative tolerances is the self-repairing. Metagenomic analysis already revealed the repairing system is enriched in Methanocellales (Erkel et al. 2006). The analysis of pure culture genomes expanded these mechanisms with more details (Lyu and Lu 2018). ROS once formed can cause extensive damages to cell components. For instances, DNA mutation or dysfunction may occur due to the oxidation of purines and pyrimidines (Dalhus et al. 2009). The membrane lipids can be oxidized into phospholipid hydroperoxides (PLOOH). The proteins containing sulfur amino acids can be deformed with the formation of disulfide bonds or methionine sulfoxide, leading to disorder of protein structures (Manevich et al. 2002). In addition, ROS may disrupt the iron-sulfur (FeS) clusters which are the prosthetic groups of many enzymes in methanogens. Genes coding for DNA base repairing and S–S or S=O group-reducing enzymes were moderately or strongly enriched in Methanocella compared with the Class I methanogens (Lyu and Lu 2018). The enrichment of cytoplasmic S–S reduction enzymes in Methanocella is consistent with the elevated abundance of thioredoxins relative to other hydrogenotrophic methanogens. The genes coding for PLOOH reduction (peroxiredoxins) are also enriched in Methanocella.

7 Conclusive Remarks

Methanocellales represent a novel type of methanogens initially discovered with DNA fingerprinting of paddy soils. These methanogens were often detected in rice rhizosphere or in association with rice roots. Earlier studies demonstrated that a considerable fraction of the plant-photosynthesized C is allocated to rice roots, released into rice rhizosphere and thereby the DOC pool serves as a major carbon source for methanogenesis, leading to the seasonal maxima of CH4 emissions. Strikingly, methanogenesis appears to occur close to rice roots. This methanogenic activity was not very expected because rice plants have a well-developed aerenchyma system where O2 can diffuse from the plant top to roots and released into the rhizosphere. As a result, rice roots and rhizosphere are partly oxic. The dilemma of active methanogenesis in the rice rhizosphere and the nature of strictly anaerobic lifestyle of methanogens causes a huge curiosity to look into the biological logic and mechanism.

Due to the nature of difficult-to-cultivation, intensive studies using molecular techniques were conducted with a focus on the ecophysiology of methanogens in paddy soils. Meanwhile, multiple efforts for enrichment and cultivation were undergone. Strikingly, molecular techniques including DNA-SIP approach revealed that albeit as hydrogenotrophic methanogens Methanocellales dominated over other methanogens when H2 partial pressure was low, indicating that out of the vast methanogenic populations in paddy soil Methanocellales might be better adapted under low H2 condition. This trait is possibly a reason why they escaped the isolation albeit existing widespread in environments. The exploration under in situ conditions using RNA-SIP technology revealed that Methanocellales play the key role in CH4 production in rice rhizosphere. Further studies were then focused on why they can adapt to low H2 condition and thrive in the rhizosphere where O2 leaks can occur.

Metagenomic investigation revealed a series of traits that support the adaptation of Methanocellales to rhizosphere environment. They possess multiple sets of antioxidant systems and repair systems. They are versatile to assimilate various sources of N and S and they may activate different core metabolisms to facilitate biosynthesis and survival during environment shift to oxic conditions. After continuous efforts for years, three strains were finally isolated into pure culture. Extensive genomic analyses were conducted to reveal the taxonomic, evolutional, and ecological properties of Methanocellales. The phylogenetic analyses using multiple marker genes in combination with genome alignment and AAI analyses consistently suggest that M. conradii and M. paludicola are closely related each other and together can be classified as a new genus while M. arvoryzae may belong to another genus. Comparative genomic analyses reveal that metabolic features for Methanocellales appear to be more diverse than previously predicted from metagenomic investigation. Three strains share close resemblance as well as novel features on the core metabolisms, such as specialization in utilizing H2 at low concentrations. For the adaptation to oxic condition that is key for their activity in rice rhizosphere, at least three general evolutionary mechanisms have been acquired and enriched in Methanocellales. The first is the usage of enzymes producing less ROS in the central methanogenesis pathway, particularly the flavin-based electron bifurcation system has been modified from classical hydrogenotrophic methanogens toward a less possibility of ROS production. The second is the expansion and diversification upon a core antioxidant system for the O2/ROS elimination. And the third is the occurrence of multiple self-repairing pathways from O2/ROS damages. Further studies are necessary to explore these novel genomic features, which would not only contribute to a deeper understanding of Methanocellales and methanogens in general but pave a way towards the development of biotechnology for control of methane emissions from paddy fields.