International Journal of Systematic and Evolutionary Microbiology (2008), 58, 294–301 DOI 10.1099/ijs.0.

65394-0

Methanolinea tarda gen. nov., sp. nov., a methane-

producing archaeon isolated from a methanogenic
digester sludge
Hiroyuki Imachi,1,2 Sanae Sakai,1 Yuji Sekiguchi,1,3 Satoshi Hanada,3
Yoichi Kamagata,1,3,4 Akiyoshi Ohashi1 and Hideki Harada1,5
Correspondence 1
Department of Environmental Systems Engineering, Nagaoka University of Technology, Nagaoka,
Hiroyuki Imachi Niigata 940-2188, Japan
imachi@jamstec.go.jp 2
Subground Animalcule Retrieval (SUGAR) Program, Extremobiosphere Research Center, Japan
Agency for Marine-Earth Science & Technology (JAMSTEC), Yokosuka, Kanagawa 237-0061,
Japan
3
Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science
and Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan
4
Research Institute of Genome-Based Biofactory, National Institute of Advanced Industrial Science
and Technology (AIST), Sapporo, Hokkaido 062-8517, Japan
5
Department of Civil Engineering, Tohoku University, Sendai 980-8579, Japan

A novel methane-producing archaeon, strain NOBI-1T was isolated from an anaerobic,

propionate-degradation enrichment culture, which was originally obtained from a mesophilic
methanogenic sludge digesting municipal sewage sludge. Cells were non-motile, rod-shaped,
0.7–1.0 mm by 2.0 mm, and formed multicellular filaments longer than 8 mm. Growth was
observed between 35 and 55 6C (optimum 50 6C) and pH 6.7 and 8.0 (optimum pH 7.0). The
G+C content of the genomic DNA was 56.3 mol%. The strain utilized H2 and formate for growth
and methane production. Based on comparative sequence analyses of the 16S rRNA gene
and mcrA gene (encoding the alpha subunit of methyl-coenzyme M reductase, a key enzyme in the
methane-production pathway), strain NOBI-1T was affiliated with the order Methanomicrobiales,
but it was significantly distant from any other known species within the order. The most closely
related species based on 16S rRNA and mcrA gene sequence similarity were respectively
‘Candidatus Methanoregula boonei’ (93.7 % 16S rRNA gene sequence similarity) and
Methanocorpusculum parvum (74.2 % deduced McrA amino acid sequence similarity to the type
strain). These phenotypic and genetic properties justified the creation of a novel species of a new
genus for the strain, for which we propose the name Methanolinea tarda gen. nov., sp. nov.
The type strain of Methanolinea tarda is strain NOBI-1T (5DSM 16494T 5JCM 12467T 5NBRC
102358T).

Methane-producing archaea (methanogens) are one of the
key populations in methanogenic waste and wastewater
treatment processes because they are responsible for the
Abbreviation: FISH, fluorescence in situ hybridization.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene
sequence of strain NOBI-1T is AB162774 (other 16S rRNA gene clonal
sequences have been deposited as accession numbers AB233309,
AB233010 and AB233299–AB233302). The GenBank/EMBL/DDBJ
accession numbers for the mcrA gene sequences of strains NOBI-1T
and SANAE are respectively AB300466 and AB300467.
Details of 16S rRNA gene phylotypes obtained from the sludge,
information on relative abundance of various groups of organisms and a
graph of methane production by strain NOBI-1T are available as
supplementary material with the online version of this paper.
final step of the degradation of organic substances
(Anderson et al., 2003; Garcia et al., 2000). So far, a
number of methanogens, placed in a wide range of
taxonomic groups within the phylum Euryarchaeota, have
been isolated from various methanogenic treatment
processes and characterized as novel species belonging to
genera such as Methanobacterium, Methanoculleus and
Methanosaeta (Garcia et al., 2000; Garrity & Holt, 2001). In
addition, recent molecular surveys targeting the 16S rRNA
gene and mcrA gene, encoding the alpha subunit of methyl
coenzyme M reductase, have revealed that numerous
unidentified methanogens may also exist in such ecosys-
tems (Chouari et al., 2005; Sekiguchi & Kamagata, 2004;
Sekiguchi, 2006; Shigematsu et al., 2004).

294 65394 G 2008 IUMS Printed in Great Britain


Recently, we reported the isolation of a novel methano-
genic archaeon, designated strain NOBI-1T, from a
mesophilic, methanogenic sludge digesting municipal
sewage sludge (Sakai et al., 2007). To cultivate the strain,
we used the ‘co-culture’ method, which takes advantage of
low but predictable H2 evolution rates from a companion
syntrophic bacterium to enrich and isolate the methano-
gen. The point was that the co-culture method mimics the
in situ substrate (H2) flow in natural environments by
adding a propionate-oxidizing H2-producing syntroph and
propionate as the H2 source for methanogens. In the
previous study, we described the isolation of the strain in
pure culture, but we did not characterize the strain fully. In
this report, we describe the detailed isolation procedure,
morphological and physiological characteristics and genetic
features of strain NOBI-1T and propose this strain as a
member of a novel species of a new genus.
A methanogenic sludge sample was obtained from a
municipal sewage plant at Nagaoka, Niigata, Japan. After
sampling, the sludge was washed immediately with phos-
phate buffer (10 mM, pH 7.2) and homogenized briefly to
inoculate into a primary enrichment culture medium. The
medium for cultivation of strain NOBI-1T was prepared as
described previously (Sekiguchi et al., 2000). Enrichment
cultures were incubated anaerobically at 37 uC. After
isolation of the strain, all axenic incubations were performed
at 50 uC in 50 ml serum vials containing 20 ml medium (pH
at 25 uC, 7.2) under an atmosphere of N2/CO2 (80 : 20, v/v)
without shaking, unless otherwise mentioned. The serum
vials were sealed with butyl rubber stoppers and aluminium
crimp seals. To monitor whether the media were kept under
anaerobic conditions, resazurin was added to the medium as
a redox indicator. In the nutritional tests, substrates added
to the vials were neutralized prior to inoculation. Growth
and substrate utilization were determined by monitoring
turbidity by optical density at 600 nm and the production of
methane. All incubations for growth/substrate utilization
tests were performed at 50 uC for over 3 months. During the
incubation for 3 months, all media had been kept under
anaerobic conditions (since the resazurin remained clear in
colour). Effects of pH, temperature and NaCl concentration
on growth of strain NOBI-1T were determined in medium
containing 0.01 % yeast extract and 1 mM acetate in the
presence of approx. 150 kPa H2. For determination of the
optimum pH for growth, the pH of the hydrogen-
supplemented medium was adjusted at room temperature
to 5.5–8.0 by adding HCl or NaOH solution under a 100 %
N2 atmosphere prior to inoculation. To determine the
temperature range for growth, cultures were incubated at
25–60 uC (pH 7.2). To evaluate the effect of NaCl
concentration on growth, an autoclaved NaCl solution was
added to the medium to give final concentrations of 1–30 g
l21. Effects of antibiotics on growth were evaluated with a
culture (0.01 % yeast extract, 1 mM acetate, 150 kPa H2)
supplemented with each antibiotic at the final concentration
of 100 mg ml21. All measurements were performed in
triplicate.
http://ijs.sgmjournals.org
Methanolinea tarda gen. nov., sp. nov.
Cell morphology was examined under a fluorescence
microscope (Olympus BX50F). The Gram-staining reac-
tion was performed by Hucker’s method (Doetsch, 1981).
The susceptibility to lysis was checked by adding SDS with
final concentrations of 0.01–1.0 %, and cell lysis was
determined by microscopic observation of cell integrity.
Transmission electron microscopy was performed with a
Hitachi H7000 transmission electron microscope as
described previously (Hattori et al., 2000). Short-chain
fatty acids, alcohols, methane, H2 and carbon dioxide were
measured as described previously (Imachi et al., 2000,
2002, 2006). The G+C content of the genomic DNA was
determined as described by Kamagata & Mikami (1991).
All procedures for DNA extraction, 16S rRNA gene-based
cloning and sequencing were reported previously (Imachi
et al., 2006). In the PCR amplification, we used the primer
pair Ar109f (Großkopf et al., 1998) and 1490R (Weisburg
et al., 1991) or EUB338* (Amann et al., 1990; Daims et al.,
1999; Hatamoto et al., 2007) and 1490R for the
construction of 16S rRNA gene-based archaeal and
bacterial clone libraries, respectively. We also used primers
Arch21F (DeLong, 1992) and 1490R to obtain the nearly
full-length 16S rRNA gene of the isolate. For PCR
amplification of the mcrA gene, we used primers ME1/
ME2 (Hales et al., 1996). Comparative 16S rRNA gene
sequence analysis was performed as described elsewhere
(Imachi et al., 2006). 16S rRNA gene sequence similarity
values were calculated using the Calculate Matrix function
of the ARB program with Jukes and Cantor correction
(Jukes & Cantor, 1969; Ludwig et al., 2004). A deduced
McrA amino acid sequence-based phylogenetic tree was
constructed by the neighbour-joining method implemented
in the ARB program with 164 amino acid positions and
percentage of acceptance mutations (PAM) distance cor-
rection. For both phylogenetic trees, bootstrap resampling
analysis for 1000 replicates was performed with the
neighbour-joining, maximum-parsimony and maximum-
likelihood methods to estimate the confidence of tree
topologies as described previously (Sekiguchi et al., 2006).
Quantitative PCR analysis was performed as described
previously (Imachi et al., 2006). For the quantitative
analysis, the following 16S rRNA gene-targeted PCR
primer sets were used: 519f and 907r (Lane, 1991;
Stubner, 2002) for the domain Bacteria, Ar109f and
Ar912r (Großkopf et al., 1998) for the domain Archaea
and NOBI109f (59-ACTGCTCAGTAACACGT-39) and
NOBI633 (59-GATTGCCAGTTTCTCCTG-39) for strain
NOBI-1T and its relatives. Primer NOBI109f was a slightly
modified version of the primer reported by Großkopf et
al. (1998) to match perfectly with the 16S rRNA gene
sequence of strain NOBI-1T. Primer NOBI633 was
designed using the ARB program (Ludwig et al., 2004).
For construction of standard templates, we used dilution
series of PCR-amplified 16S rRNA genes of Escherichia
coli strain JM109 (TaKaRa Bio Inc.), Methanobacterium
bryantii DSM 863T and strain NOBI-1T, which were
obtained with bacterial universal primer pair 8f/1490R
295
H. Imachi and others

(Weisburg et al., 1991) or the archaeal universal primer
pair Arch21F/1490R. The optimal PCR conditions were
determined by changing the Mg2+ concentration in the
PCR buffer (4 mM for 519f/907r, 3 mM for Ar109f/
Ar912r and 3 mM for NOBI109f/NOBI633) and the PCR
annealing temperature for each primer set (50 uC for
519f/907r, 52 uC for Ar109f/Ar912r and 64 uC for
NOBI109f/NOBI633). The quantitative PCR conditions
were as follows: initial denaturation at 95 uC for 2 min
followed by 40 cycles of denaturation at 95 uC for 0 s,
10 s annealing at the optimal temperatures mentioned
above and extension at 72 uC for 30, 35 and 21 s for
primer pairs 519f/907r, Ar109f/Ar912r and NOBI109f/
NOBI633, respectively. Fluorescence in situ hybridization
(FISH) was done according to the method described by
Sekiguchi et al. (1998). To detect strain NOBI-1T cells by
FISH, we used probe NOBI633 that was also used as a
primer for the quantitative real-time PCR analysis. To
evaluate the specificity of probe NOBI633, cells of
Methanobacterium bryantii DSM 863T, Methanospirillum
hungatei DSM 864T and Methanoculleus palmolei DSM
4273T were used as reference organisms. Hybridization
stringency of the probe was adjusted by changing the
formamide concentration in the hybridization buffer; the
stringent condition for the probe NOBI633 was deter-
mined to be 10 % (v/v) formamide in the hybridization
buffer. The oligonucleotide probe was labelled with Cy-3.
Our preliminary analyses of 16S rRNA gene-based cloning
and quantitative PCR revealed that the methanogenic
sludge used as the inoculum contained an uncultured
archaeal group within the order Methanomicrobiales,
possibly methanogens, as approximately 10 % of the total
archaeal population (Fig. 1, phylotype ND-3; Supple-
mentary Tables S1 and S2, available in IJSEM Online). To
cultivate the uncultured organisms, the sludge was
incubated anaerobically with 20 mM propionate. Cell
growth and methane production along with propionate
degradation in the culture occurred after 2 months of
incubation. During the cultivation, the H2 partial pressure
in the culture was determined to be in the range of 16–
28 Pa. The culture was successively transferred into fresh
medium every 60–80 days [10 % inoculum (v/v)].
Microscope observation showed that the propionate
enrichment culture after five repeated transfers contained
at least three morphologically distinct organisms: (i) blunt-
ended rod-shaped, F420-autofluorescent methanogen-like
cells, (ii) oval rods and (iii) coccoid-shaped microbes as a
minor population (Fig. 2a). These findings suggested that
propionate degradation was carried out by syntrophic
association between propionate-degrading microbes and
hydrogenotrophic methanogens. To identify the micro-
organisms present in the enrichment culture, 16S rRNA
gene-based clone analysis was performed. The analysis
indicated that all ten of the archaeal rRNA gene clones were
identical (Fig. 1; phylotype ND-Pro-Arc-1) and had the
same sequence as a phylotype recovered from the original
methanogenic sludge. The phylotype was affiliated with
the uncultured archaeal group within the order
Methanomicrobiales. On the other hand, the most frequently
retrieved bacterial phylotype (8/10 clones) was closely
related to Syntrophobacter fumaroxidans (GenBank accession
no. AB233309; 98 % sequence similarity), a mesophilic,
syntrophic propionate-oxidizing deltaproteobacterial

Fig. 1. Phylogenetic tree of the order Methanomicrobiales based on comparative analyses of 16S rRNA gene sequences,
showing the placement of the isolated methanogenic archaeon strain NOBI-1T (Methanolinea tarda). The tree was calculated
based on a distance matrix analysis of 16S rRNA gene sequences (neighbour-joining tree). Three 16S rRNA gene sequences
of organisms belonging to the class Thermoplasmata [Picrophilus oshimae KAW2/2T (GenBank accession no. X84901), clone
WCHD3-02 (AF050616) and clone pMC2A24 (AB019736)] were used to root the tree (not shown). Branching points that
supported probabilities above 90 % by all the analyses [based on 1000 replicates, estimated using neighbour-joining (NJ),
maximum-parsimony (MP) and maximum-likelihood (ML) methods] are indicated by solid circles, and nodes with open circles
indicate .70 % bootstrap probability support by the three analyses. Accession numbers are shown in parentheses. Bar, 0.1
nucleotide changes per sequence position.

296 International Journal of Systematic and Evolutionary Microbiology 58


Fig. 2. Micrographs of strain NOBI-1T. (a) Photomicrograph of
cells of strain NOBI-1T enriched in an anaerobic, propionate-
degrading enrichment culture. The arrow indicates NOBI-1T cells.
(b–c) Phase-contrast (b) and transmission electron (c) micro-
graphs of cells of strain NOBI-1T grown on H2 (approx. 150 kPa)
medium supplemented with acetate (1 mM) and yeast extract
(0.01 %). Bars, 10 mm (a, b) and 0.5 mm (c and inset).
species (Harmsen et al., 1998). The remaining bacterial
phylotype (2/10 clones) was affiliated with the phylum
Spirochaetes, and its most closely related cultured species was
http://ijs.sgmjournals.org
Methanolinea tarda gen. nov., sp. nov.
Spirochaeta stenostrepta (GenBank accession no. AB233310;
87 % sequence similarity). FISH analysis using the specific
probe NOBI633 for the archaeal phylotype also confirmed
that NOBI633-probe-reactive, rod-shaped cells were actually
present as a significant population in the culture (data not
shown). To isolate methanogenic archaeal cells axenically in
the propionate enrichment culture, we used substrates
generally utilized by methanogens, i.e. H2 (approx. 150 kPa)
and formate (40 mM). Colony isolation was at first
attempted by the roll-tube method, but the attempt was
unsuccessful. Purification of the methanogen was, therefore,
attempted by repetitive serial dilutions in liquid medium.
The highest dilution with growth (usually dilution 103 to
104) was always used for subculturing. After seven successive
transfers, we obtained a pure culture of the methanogenic
archaeon, strain NOBI-1T, in liquid medium supplemented
with H2 (Fig. 2b). The purity of strain NOBI-1T was
demonstrated by the failure to grow in the following media
at 37 and 55 uC: (i) thioglycollate medium (Difco) contain-
ing approx. 150 kPa H2/CO2 (in the head space) and 10 mM
sulphate; (ii) thioglycollate medium containing 20 mM
lactate and 10 mM sulphate; (iii) thioglycollate medium
containing 10 mM sucrose, 10 mM glucose, 10 mM cello-
biose and 10 mM xylose and (iv) AC medium (Difco).
Moreover, we also tested the purity by 16S rRNA gene-based
cloning analysis using the archaeal universal primer pair
Ar109f/1490R. Thirty clones was selected randomly and
sequenced. All 30 clonal sequences were identical and had
the same sequence as strain NOBI-1T. Also, we performed
FISH with 16S rRNA-targeted oligonucleotide probe
NOBI633 specific for strain NOBI-1T, and we confirmed
that all cells hybridized with the probe (data not shown). In
addition, we also evaluated purity by the failure to recover
bacterial 16S rRNA gene amplifications by PCR with a
universal bacterial primer pair EUB338*/1490R. The results
of these molecular surveys also indicated that the NOBI-1T
culture was axenic.
Cells of strain NOBI-1T were straight rods with blunt-ends,
non-motile, 0.7–1.0 mm wide and 2.0 mm long (Fig. 2b).
The cells often formed multicellular filaments longer than
8 mm in the syntrophic propionate-degrading enrichment
culture (Fig. 2a). Electron micrographs showed that the
strain had a sheath-like structure (Fig. 2c). The cells were
Gram-reaction negative. The cells were susceptible to lysis
with 0.1 % SDS (w/v), suggesting the presence of a
proteinaceous cell wall (Boone & Whitman, 1988).
The strain was strictly anaerobic, since its growth was
inhibited completely in the presence of trace quantities of
oxygen [0.1 and 0.2 % O2 (v/v)]. Yeast extract and acetate
were required for growth. H2 and formate (40 mM)
supported growth and methane production. In the H2
culture, we observed that H2 was converted to methane
stoichiometrically, which is explained by the equation
4H2+CO2 A CH4+2H2O (Supplementary Fig. S1). The
following substrates did not support growth and methane
production: pyruvate (20 mM), lactate (20 mM), acetate
(20 mM), propionate (20 mM), trimethylamine (10 mM),
297
H. Imachi and others
dimethylamine (10 mM), methylamine (10 mM), cyclo-
pentanol (20 mM), ethanol (5 mM), methanol (20 mM),
1-propanol (5 mM), 2-propanol (5 mM), 1-butanol
(5 mM) and 2-butanol (5 mM).
The optimum growth temperature of strain NOBI-1T was
50 uC, although the first enrichment culture was incubated
at 37 uC. No growth occurred below 30 uC or above 60 uC
over 3 months of incubation. The pH range for growth of
the strain was estimated to be pH 6.7–8.0, with an
optimum of pH 7.0. Under optimum conditions (pH 7.0,
50 uC), the specific growth rate on H2 medium was approx.
0.17 day21, which was calculated based on measurement of
optical density at 600 nm. To the best of our knowledge,
this specific growth rate is the slowest so far reported
among known methanogens. The strain was basically a
freshwater organism, but could grow at NaCl concentra-
tions up to 15 g l21. Strain NOBI-1T tolerated ampicillin,
penicillin G, vancomycin, kanamycin and streptomycin.
Rifampicin, tetracycline, monensin and chloramphenicol
inhibited cell growth completely.
The G+C content of total DNA in strain NOBI-1T was
56.3 mol%. For strain NOBI-1T, 1433 bp of the 16S rRNA
gene sequence was determined. Comparative 16S rRNA
gene sequence analysis showed that strain NOBI-1T was
affiliated with the order Methanomicrobiales (Fig. 1). The
closest relative of strain NOBI-1T was ‘Candidatus
Methanoregula boonei’ (sequence similarity 93.7 %;
Fig. 1), a recently described acidophilic hydrogenotrophic
methanogen isolated from an acidic peat bog (Bräuer et al.,
2006). In addition to the 16S rRNA gene sequence-based
analysis, we determined the sequence of part of the mcrA
gene (763 bp) and then constructed a molecular phyl-
ogenetic tree based on deduced amino acid sequences of
mcrA genes (Fig. 3). The McrA amino acid sequence-based
tree also indicated that strain NOBI-1T was a member of
the order Methanomicrobiales. However, we could not
confirm an mcrA gene-based relationship between strain
NOBI-1T and ‘Candidatus Methanoregula boonei’ because
of the lack of information about the mcrA gene of the
latter. The closest relative on the basis on the McrA amino
298
acid sequence was Methanocorpusculum parvum XIIT
(sequence similarity 74.2 %) (Zellner et al., 1987).
Based on its physiological and molecular phylogenetic
traits, strain NOBI-1T is certainly considered to be a
member of the order Methanomicrobiales. As shown in
Table 1, the members of the order have a wide variety of
phenotypic characteristics, e.g. cell morphology and
substrate usage, with the exception that methane produc-
tion from H2 is a common physiological property.
Therefore, it is difficult to distinguish the members of
the order based solely on their physiological properties and
thus molecular phylogeny is a key indicator for the
taxonomic identification of its members (Asakawa &
Nagaoka, 2003; Boone et al., 1993; Maestrojuán et al.,
1990; Rouviére et al., 1992; Spring et al., 2005; Zellner et al.,
1989, 1999).
The 16S rRNA gene-based phylogenetic analysis presented
in this study indicated that strain NOBI-1T was most
closely related to ‘Candidatus Methanoregula boonei’
(Fig. 1). The similarity value between the organisms is
93.7 %, which is in the range of genus-level differences
among described genera belonging to the order [e.g. the
differences between the genera Methanoplanus and
Methanomicobium (93.9 %) and between the genera
Methanofollis and Methanoculleus (93.5 %)]. In addition,
our isolate and ‘Candidatus Methanoregula boonei’ have
several clearly different physiological features; i.e. cell
morphology, temperature and pH range for growth and
substrate utilization (Table 1). These results justified the
creation of a new genus for strain NOBI-1T.
Based on these phenotypic and phylogenetic findings, we
propose strain NOBI-1T as a member of a novel genus and
species with the name Methanolinea tarda gen. nov., sp. nov.
Description of Methanolinea gen. nov.
Methanolinea [Me.tha.no.li.ne9a. N.Gr. n. methane (from
N.Gr. n. meth(yl) and chemical suffix -ane) methane; L.
fem. n. linea line; N.L. fem. n. Methanolinea a methane-
producing, line-shaped morphotype].
Fig. 3. Phylogenetic tree of deduced McrA
amino acid sequences showing the relation-
ship of Methanolinea tarda NOBI-1T with
related methanogenic archaea. The tree was
constructed based on distance matrices (164
amino acid positions; PAM distance correc-
tion) by the neighbour-joining method. The
sequence of Methanobacterium bryantii
M.o.H.T was used as the outgroup reference.
Bootstrap support (.50 % indicated only) was
obtained from NJ/MP/ML methods based on
1000 replicates. Accession numbers are
shown in parentheses. Bar, 10 % estimated
sequence divergence.
International Journal of Systematic and Evolutionary Microbiology 58
 Methanolinea tarda gen. nov., sp. nov.

Table 1. Differential characteristics between strain NOBI-1T and related organisms in the order Methanomicrobiales
Strains: 1, Methanolinea tarda gen. nov., sp. nov. NOBI-1T (data from this study); 2, ‘Candidatus Methanoregula boonei’ 6A8 (data from Bräuer et
al., 2006); 3, Methanomicrobium mobile BPT (Paynter & Hungate, 1968); 4, Methanoplanus limicola M3T (Wildgruber et al., 1982); 5,
Methanogenium cariaci JR1T (Romesser et al., 1979; Maestrojuán et al., 1990; Boone et al., 1993); 6, Methanoculleus borgensis MS2T (Ollivier et al.,
1985; Maestrojuán et al., 1990); 7, Methanofollis tationis Chile 9T (Zabel et al., 1984; Boone et al., 1993; Zellner et al., 1999); 8, Methanospirillum
hungatei JF-1T (Ferry et al., 1974). ND, No data available. Numbers in parentheses for the optimum temperature, pH and NaCl concentration
indicate the range allowing growth.

Characteristic 1 2 3 4 5 6 7 8

Cell morphology Rod Rod* Rod Angular, Highly irregular Irregular Irregular Sheathed
crystal-like plate cocci cocci cocci rod
or disc-shaped
Cell width (mm) 0.7–1.0 0.2–0.3 0.7 1.5 1.0–3.0 1–2 1–2.5 0.5
Cell length (mm) 2–8D 0.8–3.0 1.5–2.0 1.6–2.8 1.0–3.0 1–2 1–2.5 7.4–.100
G+C content 56.3 ND 48.8d 47.5§ 51.6d 59d 54§ 45
(mol%)
Optimum 50 (35–55) 37 (10–40) 40 (30–45) 40 (17–41) 20–25 37 (25–55) 37–40 (25–45) 30–37
temperature (uC)
Optimum pH 7 (6.7–8.0) ~5 (4.0–5.8) 6.1–6.9 (5.9–7.7) 6.5–7.5 6.8–7.3 (6.0–8.3) 6.7 (5.5–8.0) 7 (6.6–8.8) 6.6–7.4
Optimum NaCl 0 (0–15) ND ND 10 (4–54) 26.9 ¡10 8–12 (0–70) ND
(g l21)
Motility 2 ND + + 2 2 + +
Substrate utilization
Formate + 2 + + + + + +
2-Propanol/CO2 2 ND 2 2 + + 2 2
Growth requirements
Yeast extract + + + 2 + 2 + ND
Acetate + + + + + + + ND

*Cells sometimes become spherical with a diameter of 0.3–0.8 mm.

DCells often form multicellular filaments longer than 8 mm in the syntrophic propionate-degrading enrichment culture.
dDetermined by buoyant density.
§Determined by thermal denaturation.

Rod-shaped cells with blunt-ends. Often form multicellular
filaments. Methanogenic and strictly anaerobic members of
the order Methanomicrobiales, phylum Euryarchaeota,
domain Archaea. Can use H2 or formate for growth and
methane production. The type species is Methanolinea tarda.
Description of Methanolinea tarda sp. nov.
Methanolinea tarda (L. fem. adj. tarda slow, referring to its
slow growth).
Strictly anaerobic. Cells are non-motile and rod-shaped,
0.7–1.0 mm wide and 2.0 mm long. Cells often form
multicellular filaments longer than 8 mm in syntrophic
propionate-degrading enrichment culture. Gram-reaction
negative. H2 and formate can be used for growth and
methane production. Yeast extract and acetate are required
for growth. The temperature range for growth is 35–55 uC
(optimum 50 uC). The pH range for growth is 6.7–8.0
(optimum pH 7.0). Under optimum conditions, the
growth rate is approx. 0.17 day21. Growth occurs in the
presence of 0–1.5 % NaCl but does not occur in the
presence of 2.0 % NaCl. The G+C content of the DNA of
the type strain is 56.3 mol%.
The type strain, NOBI-1T (5DSM 16494T 5JCM 12467T
5NBRC 102358T), was isolated from a mesophilic,
methanogenic sludge digesting sewage sludge.
Acknowledgements
We thank Xian-Ying Meng and Mizuho Muramatsu at the National
Institute of Advanced Industrial Science and Technology (AIST) for
transmission electron microscopy and determination of DNA base
content, respectively. This research was supported by grants from the
Japan Society for the Promotion of Science, the New Energy and
Industrial Technology Development Organization and the Institute
for Fermentation, Osaka.
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