Keywords

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Taxonomy, Historical and Current

Short Description of the Family

Methanocellaceae (Me.tha.no.cel.la′ce.ae N.L. fem. n. Methanocella type genus of the family; -aceae the ending to donate a family; N.L. fem. pl. n. Methanocellaceae the family of the genus Methanocella).

Phylogenetically, the family Methanocellaceae is placed in the order Methanocellales within the phylum Euryarchaeota (Sakai et al. 2008). The family contains a single genus Methanocella, which consists of three species: Methanocella paludicola, Methanocella arvoryzae, and Methanocella conradii (Sakai et al. 2008, 2010; Lü and Lu 2012a). Cells occur singly and almost all of the cells are rod-shaped. In some species, coccoid cells were observed in late-exponential culture. The cells are nonmotile. Methane is produced from H2/CO2 or formate. Acetate is required as a carbon source for growth.

Phylogenetic Structure of the Family and Its Genus

The family Methanocellaceae is a single family within the order Methanocellales. According to phylogenetic analysis based on the 16S rRNA gene, the order Methanocellales deeply branches between other orders of methanogens, Methanosarcinales and Methanomicrobiales (Fig. 12.1). Phylogenetic analysis of the mcrA gene (encoding the subunit of methyl-coenzyme M reductase, a key enzyme in the methane production pathway) also indicates that the order Methanocellales is distinct from the orders Methanosarcinales and Methanomicrobiales (Sakai et al. 2008).

Fig. 12.1
figure 03181figure 03181

Neighbor-joining phylogenetic tree showing the placement of the family Methanocellaceae based on 16S rRNA gene sequences. The tree was constructed by using a subset of sequences representative of closely related genera to stabilize tree topology. In addition, a 40 % conservational filter for the whole archaeal domain was used to remove hypervariable positions. The type species of the family is indicated in bold. The bar represents the number of changes per sequence position

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Molecular Analyses

Genome Comparison

Genome sequences are available for all three species of the family Methanocellaceae (Erkel et al. 2006; Sakai et al. 2011; Lü and Lu 2012b).

The genome of M. paludicola SANAET is a single circular chromosome of 2,957,635 bp length with a GC content of 54.9 %. Two clusters of rRNA operons and two more distantly located 5S rRNA genes are present in the genome. A total of 48 tRNA genes containing putative introns are scatted over the genome. 3,004 predicted protein-coding sequences (CDSs) were identified. 1,467 genes (48.8 % of the protein-coding genes) were assigned with a putative function while the remaining ones (1,537 genes; 51.2 % of the protein-coding genes) were annotated as hypothetical proteins.

The genome of M. arvoryzae MRE50T is a circular chromosome with 3,179,916 bp length and a GC content of 54.6 %. There are 3,085 coding genes, three rRNA operons, and 54 tRNAs in the chromosome. Originally, the genome sequence was reported in 2006 as an environmental genotype by metagenomic analysis of a methanogenic consortium (Erkel et al. 2006). Later, M. arvoryzae MRE50T was isolated from the methanogenic consortium (Sakai et al. 2010), and the sequences of the 16S rRNA and mcrA genes obtained from strain MRE50T perfectly matched those of the complete genome sequence. So, the genome sequence based on metagenomic analysis was considered to be the true genome sequence of M. arvoryzae MRE50T.

M. conradii HZ254T has so far the smallest circular chromosome of 2,378,438 bp length among the members of the family Methanocellales. The GC content of 52.7 % is the lowest among the genomes of the family Methanocella. The genome encodes two rRNA operons and a full complement of tRNA genes. 2,512 candidate genes were identified, of which 71.9 % could be assigned a possible function.

All three genomes contain sufficient genes to encode a full methanogenesis pathway using H2/CO2. Similarly to other obligate hydrogenotrophic methanogens, formate dehydrogenase complexes and a formate transporter, the key enzyme for the growth on formate as an alternative methanogenic substrate, were also found. No homologous genes for alcohol dehydrogenase, which is involved in methanogenesis from primary or secondary alcohols, were found. Although an incomplete pathway of potential methanogenesis from methanol was found in the genome of M. arvoryzae MRE50T, none of the corresponding genes for utilizing methanol and other C1 compound were found in the M. paludicola SANAET and M. conradii HZ254T genome. The ANI (average nucleotide identity) values among the three strains were between 69.4 % and 74.8 % (Lü and Lu 2012b).

Phenotypic Analyses

The main features of members of the family Methanocellaceae are listed in Table 12.1.

Methanocella (Sakai et al. 2008)

  • Me.tha.no.cel′la. N.Gr. n. methane (from N.Gr. n. meth(yl) and chemical suffix -ane) methane; L. fem. n. cella a room, and in biology a cell; N. L. fem. n. Methanocella a methane-producing cell.

  • Cells are nonmotile rods and occur singly. The cells autofluoresce under epifluorescence microscopy when excited with light near 420 nm in wavelength, which indicates the presence of the methanogen-specific coenzyme F420. Methane is produced from H2/CO2 or formate. Acetate is required for growth. Some species also require yeast extract for its growth. The type species is Methanocella paludicola.

Table 12.1 Morphological and physiological characteristics of the species within the family Methanocellaceae
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List of Species of the Genus Methanocella

Methanocella paludicola (Sakai et al. 2008)

  • (pa.lu.di′co.la. L. n. palus -udis swamp, muddy environment; L. suff. -cola derived from L. n. incola inhabitant, dweller; N.L. masc. n. paludicola an inhabitant of muddy environments).

  • Type strain is SANAET (=JCM 13418 = NBRC 101707 = DSM 17711), which was isolated from a Japanese rice field soil. Cells occur singly and almost all of the cells are rod-shaped (1.8–2.4 mm long by 0.3–0.6 mm wide); however, coccoid cells were occasionally observed in late-exponential culture. Cells are nonmotile; flagella-like structures were not observed. Cells stain Gram-negative and are resistant to lysis by 2 % (w/v) SDS. Freeze-etched preparations indicated that cells of strain SANAET are surrounded by an S-layer with hexagonal symmetry. Colonies in a deep agar medium are white to creamy, reaching a diameter of 1–1.5 mm after 6 months incubation with H2/CO2 as a substrate. Methane is produced from H2/CO2 and formate. Acetate and yeast extract are required for growth. L-glutamate can be used in substitution for yeast extract. Growth occurs between 25 and 40 °C with optimum at 35–37 °C. The pH range is 6.5–7.8; optimum growth occurs at pH 7.0. Growth is observed in media containing less than 1 g NaCl L−1. The strain tolerates 100 μg mL−1 of ampicillin, penicillin G, vancomycin, kanamycin, bacitracin, and streptomycin, but not rifampicin, tetracycline, and chloramphenicol.

Methanocella arvoryzae (Sakai et al. 2010)

  • (ar.vo.ry′ za.e. L. n. arvum an arable field, cultivated land; L. n. oryza rice; N.L. gen. n. arvoryzae of a rice paddy field).

  • Type strain is MRE50T (= NBRC 105507 = DSM 22066), was isolated from a thermophilic methanogenic consortium, which was originally obtained from an Italian rice field soil. Cells occur singly and most of the cells are rods (1.3–2.8 mm long and 0.4–0.7 mm wide). Coccoid cells are also observed, especially in late-exponential phase cultures. Negative staining indicated that cells of strain MRE50T might possess flagella; in good agreement with this finding, genes putatively encoding archaeal flagella were detected in the genome (Erkel et al. 2006). However, motility has not been observed. Freeze-etched preparations indicated that cells of strain MRE50T were surrounded by an S-layer with hexagonal symmetry. Colonies in a deep agar medium are white to creamy, reaching a diameter of 0.1–1 mm after 2–3 weeks incubation with H2/CO2 as substrate. Methane is produced from H2/CO2 and formate. Acetate is required for growth and yeast extract enhances growth. Growth occurs at 37–55 °C with optimum at 45 °C. The pH range is from 6 to 7.8, optimum at pH 7.0. Growth is observed in media containing less than 20 g NaCl L−1. The strain is resistant to 100 μg mL−1 of ampicillin, vancomycin, kanamycin, rifampicin, tetracycline, and streptomycin, but not to chloramphenicol.

Methanocella conradii (Lü and Lu 2012a)

  • (con.rad′i.i. N.L. gen. masc. n. conradii, named after Ralf Conrad, who has pioneered the ecological studies on Methanocella methanogens in environmental samples).

  • The type strain is HZ254T (= CGMCC 1.5162 = JCM 17849 = DSM 24694), isolated from a Chinese rice field soil. Cells are rods (1.4–2.8 μm long and 0.2–0.3 μm wide) and occur singly. A flagellum was observed by negative staining of the cells being consistent with the detection of a fla gene cluster in the genome (Lü and Lu 2012b). However, motility has not been observed. Cells lyse in 0.5 % but not in <0.1 % SDS. Colonies are nearly lens-shaped in roll-tube medium. Methane is produced from H2/CO2, but not from formate. Acetate is required for growth and yeast extract can stimulate growth. Growth occurs at 37–60 °C (optimum 55 °C), at pH 6.4–7.2 (optimum 6.8) and with less than 5 g L−1 of NaCl (optimum 0–1 g L−1). The strain tolerates 200 μg mL−1 of ampicillin, penicillin G, and kanamycin, but not apramycin, neomycin, rifampicin, and chloramphenicol.

Isolation, Enrichment, and Maintenance Procedures

Enrichments and isolation must be carried out under anaerobic conditions. A selective enrichment culture procedure has not yet been developed. However, cultivation under low hydrogen concentration, called co-culture method (Sakai et al. 2007, 2009), which is achieved by using an anaerobic, syntrophic, heterotrophic, and hydrogen-producing bacterium as partner organism, might be effective for enrichment of Methanocella methanogens. Indeed, M. paludicola SANAET was isolated from such a culture using propionate as heterotrophic substrate (Sakai et al. 2007). Likewise cultivation of Methanocella members was reported in other syntrophic cultures (Sakai et al. 2009). In addition, incubation under moderately high temperature (e.g., 45–50 °C) with high concentration of hydrogen may also result in enrichment cultures with a high population density of Methanocella species (Fey et al. 2001; Lü and Lu 2012a). Therefore, high cultivation temperatures may also benefit cultivation of Methanocella members. Taken together, syntrophic culture conditions and moderately high incubation temperatures might be a good strategy to cultivate thermophilic species. For example, Methanocella species were predominant in syntrophic acetate-degrading enrichment cultures at 50 °C (Liu and Conrad 2010; Rui et al. 2011).

Once enrichment cultures are obtained, purification can be accomplished by repeated serial dilution with both liquid and solid media supplemented with H2/CO2 (ca. 150 kPa) or formate (40 mM). Since Methanocella species have limited growth factor requirement (Table 12.1), isolation media should better be supplemented with acetate and/or yeast extract. The addition of antibiotics may be beneficial to inhibit growth of contaminating bacteria, and allow purification of the methanogen from the enrichment culture.

M. paludicola SANAET was isolated from a rice paddy soil at Nagaoka, Niigata, Japan. Enrichment of M. paludicola SANAET was achieved by application of the co-culture approach with Syntrophobacter fumaroxidans (Harmsen et al. 1998) as the hydrogen-producing syntrophic partner (Sakai et al. 2009). A primary enrichment culture was made from the rice paddy soil with propionate (20 mM) as the sole energy source and the addition of pregrown cells of a S. fumaroxidans culture. The cultivation was carried out at 37 °C under an atmosphere of N2/CO2 (80/20, v/v) without shaking. Once cells multiplied and propionate degradation and methane production were observed, the culture was successively transferred to fresh medium every 50–80 days. The co-culture enrichment with S. fumaroxidans and Methanocella members was established after five transfers. M. paludicola SANAET was isolated from the co-culture enrichment into pure culture by applying the serial dilution method with liquid media under H2/CO2 (ca. 150 kPa) atmosphere.

M. arvoryzae MRE50T was isolated from a methanogenic consortium named MRE50, which was originally established in the year 2000, using Italian rice field soil as inoculum (Lueders et al. 2001). Isolation of M. arvoryzae MRE50T was performed at 45 °C using serial dilution method with liquid media under H2/CO2 and addition of streptomycin and vancomycin (each 100 μg mL−1) to prevent growth of bacteria. Once contaminated bacteria were eliminated, the culture was transferred to fresh liquid medium supplemented with H2/CO2 and 1 mM acetate. Thereafter, the deep agar method under H2/CO2 growth condition was applied. Colony formation was observed only after 2–3 weeks of incubation. Colonies of strain MRE50T were white to creamy in color and had a diameter of 0.1–1 mm. The deep agar isolation procedure was repeated twice.

M. conradii HZ254T was isolated from a rice field soil located in Hangzhou, China. Enrichment of strain HZ254T was performed at 50 °C under ca. 150 kPa H2/CO2 (80/20, v/v) except that the preincubation was performed under an atmosphere of N2. After at most 13 successive transfers over 338 days, the archaeal populations consisted exclusively of Methanocella species. Isolation was carried out using the roll-tube technique with a medium containing 1.5 % agar supplemented with 0.05 % yeast extract and tryptone and 1 mM acetate under a H2/CO2 atmosphere. Nearly lens-shaped colonies were formed after 5 months of incubation. The colonies were picked with Pasteur pipettes and further purified by serial dilution in liquid medium supplemented with 200 μg mL−1 kanamycin.

Cultures can be stored at room temperature for short-term preservation, at 4 °C for longer storage. It was shown that a culture was able to recover after 502 days storage at 4 °C (Lü and Lu 2012a). Cultures can also be preserved at −80 °C in liquid medium plus 15 % (v/v) glycerol.

Ecology

The main habitat of Methanocella members appears to be soil, especially rice field soil, since all species of the genus Methanocella have so far been isolated from rice field soil. In addition, 16S rRNA gene-based analyses indicate that Methanocella-related sequences are the major archaeal component in rice fields regardless of geographical location and season (Ramakrishnan et al. 2001; Krüger et al. 2005). In rice fields, Methanocella species colonize especially rice roots (Großkopf et al. 1998) and play a key role in CH4 production from plant-derived carbon (Lu and Conrad 2005). Methanocella members are probably one of the most important methanogenic groups responsible for CH4 emission from rice fields.

The question arises, why Methanocella members are dominant particularly in rice fields, in spite of the presence of other hydrogenotrophic methanogens, which seem to perform the same ecosystem function? The ecological specialty of Methanocella members was demonstrated by using the stable-isotope-probing technique. In situ pulse labeling of rice plants with 13CO2 demonstrated that mainly Methanocellaceae living in the rhizosphere were assimilating the plant-derived carbon while producing methane (Lu and Conrad 2005). Labeling the archaeal communities on excised rice roots with 13CO2 showed that 13C was preferentially incorporated into Methanocella members when the incubation was done under a N2 atmosphere in which low concentrations of H2 were generated by fermentative bacteria living from rice root materials (Lu et al. 2005). However, Methanobacteriales or Methanosarcinales incorporated the 13C when the rice roots were incubated under high hydrogen concentrations (Lu et al. 2005). Stable-isotope-probing analysis with 13C-labeled propionate of anoxic rice field soil (Lueders et al. 2004) showed that 13C was incorporated not only into rRNA of syntrophic propionate-oxidizing bacteria but also into methanogenic archaea, including Methanocella members. In methanogenic environments, the oxidation of propionate requires the syntrophic cooperation of propionate-oxidizing bacteria with hydrogenotrophic methanogens due to thermodynamic constraints. Therefore, propionate degradation can only proceed if hydrogen concentrations are kept low. Collectively, these results indicate that Methanocella members may have an ecological niche characterized by low H2 concentrations, whereas other methanogens are active when H2 concentrations are high. This interpretation is consistent with the successful cultivation and isolation of Methanocella members using the co-culture method (Sakai et al. 2007, 2009). Although affinity data for hydrogen of Methanocella members have not yet been reported, we postulate that they must have a comparatively high affinity for hydrogen. This may explain why Methanocella members are dominant species in rice fields, where hydrogen concentration is low (Conrad et al. 1986, 1989).

An additional characteristic of the Methanocella methanogens may be their moderately thermophilic mode of activity. Although rice field soils have a relatively low temperature regime (about 15–30 °C, Schütz et al. 1990), the presence of thermophilic methanogens was demonstrated in several studies. In fact, the methanogenic community in rice field soil produces CH4 over a wide temperature range up to about 50 °C (Yao and Conrad 2000; Fey et al. 2001) and moderately thermophilic methanogens, mostly Methanocellaceae are ubiquitously present (Wu et al. 2006). Temperature increase was found to affect not only the rate of CH4 production but also the composition of the methanogenic community and its function. Conrad et al. (2009) reported that when rice filed soil was incubated at moderate temperatures (<40 °C), CH4 is always produced by a combination of aceticlastic and hydrogenotrophic methanogenesis involving Methanosarcinales, Methanomicrobiales, Methanobacteriales, and Methanocellales, while at moderately high temperatures (>40 °C), CH4 is formed exclusively by hydrogenotrophic methanogenesis with Methanocellales prevailing. Stable-isotope-probing analysis with 13C-labeled acetate at 50 °C demonstrated that the bacterial community consisted mainly of members of the Thermacetogenium genus, i.e., thermophilic syntrophic acetate oxidizers, while the archaeal community consisted mainly of Methanocella members (Liu and Conrad 2010). Hence, Methanocella members are probably involved in syntrophic acetate oxidation at thermal conditions. The moderately thermophilic characteristics of Methanocella members helped to isolate two of the existing pure cultures of Methanocella species (Sakai et al. 2010; Lü and Lu 2012a). However, all these results were obtained from artificially incubated rice field soils. The selective advantage of moderate thermophily in real environments is presently unknown.

Finally, Methanocellaceae seem to have a rather high resistance to aeration. Initially, this was hypothesized because of the occurrence in the genome of many different genes involved in detoxification of oxygen species, including catalase and hyperoxide dismutase (Erkel et al. 2006; Lü and Lu 2012b). Such resistance is beneficial for methanogens inhabiting rice fields because of regular drainage and fallow under non-flooded and oxic conditions. Indeed, it was found that drainage and aeration does not impede methanogenic activity in rice field soil and that populations of methanogens are maintained at the same titer as under flooded and anoxic conditions (Krüger et al. 2005; Watanabe et al. 2009; Yuan et al. 2011; Ma et al. 2012). An intriguing observation was the detection of Methanocellaceae in dry upland soils, even in biological soil crusts of desert, which are most of the time under oxic conditions. In such soils, hydrogenotrophic Methanocellaceae together with presumably aceticlastic Methanosarcinaceae are the only methanogenic archaea. They can rapidly be activated to produce CH4 and proliferate when the soil is wetted (Angel et al. 2011, 2012a, b). These observations indicate that Methanocellaceae are characterized by being able to tolerate oxic conditions and desiccation.

While all existing Methanocella species were isolated from rice fields and many of the studies involving Methanocellaceae were done in rice fields, Methanocellaceae have also been detected in a variety of other anaerobic environments by using 16S rRNA gene clone sequencing. These environments include acidic peatlands, acidic bog, rich minerotrophic fen, oil-contaminated groundwater, tropical rainforest, gold mine, lake sediment, river bank soil, and bromeliad tanks (Dojka et al. 1998; Zepp Falz et al. 1999; Jurgens et al. 2000; Donovan et al. 2004; Kaku et al. 2005; Cadillo-Quiroz et al. 2006, 2008, 2010; Martinson et al. 2010; Goffredi et al. 2011). Methanocella species apparently contribute to methane production not only in rice fields but also other anaerobic environments.