Isolation and Characterization of Methanothermobacter crinale sp. nov., a Novel Hydrogenotrophic Methanogen from the Shengli Oil Field^,

Abstract

Syntrophic acetate oxidation coupled with hydrogenotrophic methanogenesis is an alternative methanogenic pathway in certain thermophilic anaerobic environments such as high-temperature oil reservoirs and thermophilic biogas reactors. In these environments, the dominant thermophilic methanogens were generally related to uncultured organisms of the genus Methanothermobacter. Here we isolated two representative strains, Tm2^T and HMD, from the oil sands and oil production water in the Shengli oil field in the People's Republic of China. The type strain, Tm2^T, was nonmotile and stained Gram positive. The cells were straight to slightly curved rods (0.3 μm in width and 2.2 to 5.9 μm in length), but some of them possessed a coccal shape connecting with the rods at the ends. Strain Tm2^T grew with H₂-CO₂, but acetate is required. Optimum growth of strain Tm2^T occurred in the presence of 0.025 g/liter NaCl at pH 6.9 and a temperature of 65°C. The G+C content of the genomic DNA was 40.1 mol% ± 1.3 mol% (by the thermal denaturation method) or 41.1 mol% (by high-performance liquid chromatography). Analysis of the 16S rRNA gene sequence indicated that Tm2^T was most closely related to Methanothermobacter thermautotrophicus ΔH^T and Methanothermobacter wolfeii VKM B-1829^T (both with a sequence similarity of 96.4%). Based on these phenotypic and phylogenic characteristics, a novel species was proposed and named Methanothermobacter crinale sp. nov. The type strain is Tm2^T (ACCC 00699^T = JCM 17393^T).

INTRODUCTION

Methanogenesis is the terminal process of organic compound degradation and plays a major role in the global carbon cycle, occurring in a variety of natural and artificial environments, such as the gastrointestinal tracts of animals, rice paddy soils, deep subsurface marine or freshwater sediments, and anaerobic bioreactors (14, 28). The most important precursors for methane production during anaerobic digestion of organic matter are acetate and H₂-CO₂, which are converted into methane by aceticlastic and hydrogenotrophic methanogens (28), respectively. However, an alternative pathway for acetate is syntrophic acetate oxidation followed by hydrogenotrophic methanogenesis (65). Several bacteria responsible for acetate oxidation have been isolated and characterized (17, 25, 48, 60), and a series of reports revealed that this process is present in high-temperature oil reservoirs (15, 33, 40), thermophilic or mesophilic biogas reactors (16, 21, 23, 43, 49), lake sediments (41), and rice paddy soils (27). Interestingly, it is proposed that syntrophic acetate oxidation coupled to hydrogenotrophic methanogenesis is the main methanogenic pathway in high-temperature petroleum reservoirs (33) and probably involved in the thermophilic methanogenic degradation of hydrocarbon (15). Cloning of rRNA genes from these environments indicates that the dominant thermophilic hydrogenotrophic methanogens were found to be affiliated with the genus Methanothermobacter. However, to our knowledge, pure isolates have not been described. Here, we report the isolation and characterization of two strains, Tm2^T and HMD, belonging to a novel phylotype of Methanothermobacter.

MATERIALS AND METHODS

Samples and media.

The production water of oil reservoirs was collected from the Shengli oil field in 2007 and maintained in our lab at a temperature of 4°C. The oil reservoir is located 1,680 to 1,800 m below the sea floor and has a pressure of 9.24 MPa. The in situ temperature of the reservoir ranges from 75°C to 80°C, and the total salt concentration of the oil production water is 9,794 mg/liter. The oil sands comprised crude oil (30.2%), water (52.8%), and other solid sands. They were sampled from oil-water separating tanks at the Haoxian central facility of the Shengli oil field in 2003 and maintained at room temperature.

Saltwater medium containing (per liter) 20.0 g NaCl, 0.5 g MgCl·6H₂O, 0.15 g CaCl₂·2H₂O, 0.3 g NH₄Cl, 0.2 g KH₂PO₄, 0.5 g KCl, and 0.5 g l-cysteine hydrochloride (13) was used for enrichment and purification. It was autoclaved for 30 min at 121°C and then reduced with Na₂S·9H₂O (0.03%). A solution of sterile NaHCO₃ (0.25%), a vitamin solution (2 ml/liter) (34), trace element solution SL-7 (2 ml/liter) (61), vitamin B₁₂ (2 ml/liter) (13), and vitamin B₁ (2 ml/liter) (13) were injected into the medium before inoculation, and the pH was adjusted to 7.0 to 7.2. The basal medium used was composed of 0.5 g NaCl, 0.5 g MgCl·6H₂O, 0.1 g CaCl₂·2H₂O, 0.3 g NH₄Cl, 0.2 g KH₂PO₄, 0.5 g KCl, and 0.5 g l-cysteine hydrochloride (13). Modified DSM medium 119 was prepared with (per liter) 0.5 g KH₂PO₄, 0.4 g MgSO₄·7H₂O, 0.4 g NaCl, 0.4 g NH₄Cl, 0.05 g CaCl₂·2H₂O, 1.0 ml trace element solution SL-7 (61), 1.0 g yeast extract (YE), 1.0 g sodium acetate, 2.0 g sodium formate, 5.0 ml sludge fluid (prepared according to the instructions for DSM medium 119), 2.5 g NaHCO₃, 0.5 g cysteine-HCl·H₂O, and 0.5 g Na₂S·9H₂O, and the pH was adjusted to 6.7 to 7.0. Aliquots were distributed into glass vials sealed with butyl rubber stoppers and aluminum caps or transferred into anaerobic tubes. All of the media were prepared anaerobically under a gas atmosphere of 80% N₂ and 20% CO₂ via the Hungate anaerobic technique (31), in which resazurin (1 mg/liter) is a redox indicator.

Enrichment and purification.

Samples (about 10 g) of oil sands were distributed into 120-ml vials containing 50 ml saltwater medium and incubated statically at 55°C in the dark. After 118 days, the initial preenrichment with positive methane production was transferred into fresh saltwater medium and incubated statically at 55°C in the dark.

After several months of incubation, the Hungate roll tube and serial-dilution methods were applied for the isolation of pure cultures (18). Approximately 0.5-ml methanogenic enrichments were serially diluted in 5 ml of saltwater medium in roll tubes and incubated at 55°C with 1.5% melting agar amended with sodium acetate (0.1 mmol/liter), trimethylamine (0.1 mmol/liter), and a gas mixture of H₂ and CO₂ (4/1, vol/vol, 200 kPa). Fluorescent colonies (420 nm) were picked into saltwater medium with the addition of YE (0.5 g/liter), sodium acetate (0.1 mmol/liter), and trimethylamine (0.1 mmol/liter) under a gas mixture of H₂ and CO₂ (4/1, vol/vol, 200 kPa). After several repeated isolations, pure cultures were obtained and assessed with a purity test medium that contained 4.0 g beef extract, 10.0 g Trypticase peptone, 10.0 g YE (Oxoid), 4.0 g glucose, 1.0 g maltose, 1.0 g soluble starch, 5.0 g NaCl, 0.001 g resazurin, 0.5 g cysteine-HCl·H₂O, and1 liter distilled water with the pH adjusted to 7.0 to 7.5 (5). Purity tests were conducted under both anaerobic and aerobic conditions.

The other methanogenic enrichment was obtained from a 10-ml mixture of oil production water in 50 ml medium at 60°C, and a serial-dilution method was used to isolate the thermophilic methanogen under a gas mixture of H₂ and CO₂ (4/1, vol/vol, 200 kPa).

Methods of analysis.

CH₄ and CO₂ were analyzed with a gas chromatograph (Shimadzu GC 2010) with a Porapak Q column and a thermal conductivity detector. The column, oven, and detector temperatures were 50°C, 50°C, and 70°C, respectively. The carrier gas was hydrogen (99.999%) at a flow rate of 50 ml/min. Gas, 0.2 ml, was injected into the column using pressure lock syringes (Vici). The total amount of each gas was calculated based on Avogadro's law after calibration with a gas mixture of N₂, CH₄, and CO₂ at 29.96%, 39.99%, and 30.05%, respectively.

Volatile fatty acids (VFA) were analyzed by gas chromatography (GC; Shimadzu GC-7AG) with a stainless steel column packed with GDX103 (pretreatment with 5% phosphoric acid) and a flame ionization detector. The temperatures of the injection port, oven, and detector were 210°C, 180°C, and 210°C, respectively. Nitrogen was supplied as the carrier gas, and the flow rates of nitrogen, hydrogen, and air were 70, 50, and 500 ml/min, respectively. After acidification with H₂SO₄ to pH <2 and centrifugation, 10 μl of supernatant was injected for GC analysis with a data processor (Shimadzu C-R1B). A mixture of 100 ppm acetic acid, 100 ppm propionic acid, and100 ppm butyric acid was used as an external standard.

Growth and genomic characteristics. (i) Microscopy.

Cultures were regularly observed under a phase-contrast and immunofluorescence microscope (Nikon 80i). The Gram reaction was determined as described previously (2). A Hitachi 3400N scanning electron microscope was used to observe cell morphology (5); cells were negatively stained with 1% uranyl acetate and observed in a JEM 1230 transmission electron microscope (36).

(ii) Biochemical and physiological analyses.

To investigate potential substrates, sodium formate (20 mM), sodium acetate (20 mM), trimethylamine (20 mM), monomethylamine (20 mM), ethanol (20 mM), dimethyl sulfide (10 mM), isopropanol (10 mM), isobutanol (10 mM), 2-butanol (10 mM), and H₂-CO₂ (4/1, vol/vol, 200 kPa) were tested separately in triplicate in modified DSM medium 119 at 65°C in the dark.

Required growth factors were tested in basal medium at 65°C with H₂-CO₂ (4/1, vol/vol, 200 kPa) in which each component of the medium was added as follows: (i) a mixture of coenzyme M (coM; 25 mg/liter), YE (1 g/liter), and vitamins and trace element solution SL-7 (2 ml/liter) (34, 61); (ii) coM (25 mg/liter); (iii) sludge fluid (10 ml/liter); (iv) a mixture of vitamin and trace elements solutions SL-7 (2 ml/liter) (34, 61); (v) YE (1 g/liter); (vi) sodium acetate (10 mM); (vii) NiCl₂ (0.01 g/liter); and (viii) selenite-tungstate solution (0.01 ml/liter) (52). The effect of pH on strain Tm2^T was determined in basal medium amended with YE (1 g/liter) and sodium acetate (0.1 g/liter) at 65°C. The pH was adjusted from 5.5 to 9.0 at room temperature with different buffers (morpholineethanesulfonic acid for pH 6 to 6.5, CO₂-bicarbonate buffer for pH 7, Tris-HCl for pH 7.5 to 8.5, and sodium carbonate-bicarbonate buffer for pH 9) under different partial pressures of CO₂ (38 kPa for pH 6 to 7.0, 25 kPa for pH 7.5 to 8.5, and 13 kPa for pH 9.0) and a constant partial pressure of H₂ (152 kPa) in the headspace (5). The effect of temperature on strain Tm2^T was determined in basal medium amended with YE (1 g/liter) and sodium acetate (0.1 g/liter) with temperatures ranging from 40 to 85°C under a gas mixture of H₂ and CO₂ (4/1, vol/vol, 200 kPa). The effect of the NaCl concentration on strain Tm2^T was determined in basal medium amended with sodium acetate (0.1 g/liter) at a pH of 7.0 to 7.2 under a gas mixture of H₂ and CO₂ (4/1, vol/vol, 200 kPa) at 65°C. All of the experiments mentioned above were carried out statically in triplicate with 20 ml of medium (60-ml vials) which was inoculated (5%, vol/vol) with a culture in the exponential growth phase.

(iii) Susceptibility experiments.

The sensitivity of strain Tm2^T to the antibiotics ampicillin (200 mg/liter), rifampin (20 mg/liter), neomycin (20 mg/liter), chloramphenicol (20 mg/liter), apramycin (20 mg/liter), kanamycin (100 mg/liter), and penicillin G (10 mg/liter) was tested in 10 ml of basal medium amended with YE (1 g/liter) and sodium acetate (0.1 g/liter) with H₂-CO₂ (4/1, vol/vol, 200 kPa) at 65°C. Growth was determined from methane production. SDS susceptibility tests were performed with SDS concentrations of 0.01%, 0.1%, and 1% (wt/vol) (2).

(iv) G+C content determination.

Genomic DNA was isolated by SDS treatment after grinding under liquid N₂ as previously described (19), and its G+C content was determined by the thermal denaturation (T_m) method (6, 32) and the high-performance liquid chromatography (HPLC) method (35). The genomic DNA of Escherichia coli K-12 was used as a reference.

Phylogenetic analysis.

About 1.5 ml of liquid culture was collected for genomic DNA extraction by bead beating (42). DNA products were purified with the Promega Wizard DNA cleanup system (Promega) and stored at −25°C. The 16S rRNA and mcrA gene fragments were amplified using primers Ar21F/1492R, Ar109f/Ar915r, and MCRf/MCRr, respectively (8, 29, 53), reaction mixtures of 50 μl consisted of 10 to 50 ng template DNA, 5 μl 10× PCR buffer, 4 μl deoxynucleoside triphosphates (each at 2.5 mM), 3 μl MgCl₂ (25 mM), 0.25 μl TaKaRa Taq (5 U/μl), and 1 μl each of the forward and reverse primers (10 μM for the 16S rRNA gene and 50 μM for the mcrA gene). Amplification with archaeal primers Ar21F and 1492R was carried out as follows: 94°C for 4 min, followed by 30 cycles of 94°C for 1 min, 52°C for 1 min, and 72°C for 2 min, with a final extension at 72°C for 7 min. Amplification with archaeal primers Ar109f and Ar915r was described previously (42). PCR cycles of mcrA genes were as follows: an initial denaturation step of 3 min at 94°C; 45 s at 94°C, 45 s at 50°C, and 90 s at 72°C for a total of 27 amplification cycles; and finally 5 min at 72°C. The PCR product was purified with the LangGang general DNA agarose gel recovery kit (LangGang Biotech Co., Ltd.). The cloning and sequencing of 16S rRNA and partial mcrA gene fragments were conducted according to Rui et al. (46). The 16S rRNA gene sequences were checked for chimeras with the Bellerophon program of the Greengene database (9), and aligned with sequences in Ribosomal Database Project release 10 to search for the most closely related sequences (1, 7). The 16S rRNA gene sequences of the methanogenic enrichment clone were aligned with the ClustalX software (24), the distance matrices were calculated using the DNAdist software of the PHYLIP 3.69 package (12), and then the sequences were grouped into operational taxonomic units (OTUs) using the furthest-neighbor clustering algorithm of the DOTUR software with a 98% threshold (47). Phylogenetic trees were constructed and compared for consistency by using the neighbor-joining, minimum-evolution, and maximum-parsimony methods in the MEGA 4.0 software (54). Bootstrap values were calculated after 1,000 replications.

Nucleotide sequence accession numbers.

The 16S rRNA gene sequences of strains Tm2^T and HMD and the partial mcrA gene sequence of strain Tm2^T were deposited in the GenBank database with accession numbers HQ283273, HQ828065, and HQ283274, respectively. The GenBank accession numbers of methanogenic enrichment clones are HQ845184 to HQ845192.

RESULTS AND DISCUSSION

Enrichment and purification.

The initial preenrichment from oil sands with positive methane production was transferred into fresh saltwater medium (80 ml). Methane production was steady for 87 days at an average rate of 2.5 μmol/day before decreasing to approximately 0.7 μmol/day. The main VFA detected was acetate, which declined from 205 μmol at day 87 to 34 μmol after 302 days (Fig. 1), suggesting that methane was produced from acetate degradation (Fig. 1). Propionate was also detected at a low concentration. A molecular survey of the archaeal 16S rRNA gene clone library constructed from the first transfer of methanogenic enrichment at 302 days revealed that the archaeal community consisted of two groups OTU1 (6 clones) and OTU2 (3 clones), which were affiliated with the type strains of Methanothermobacter wolfeii DSM 2970^T (95.7% sequence similarity, AB104858) and Methanolinea tarda NOBI-1^T (81.5% sequence similarity, AB162774), respectively. The enrichment was serially diluted in 5 ml saltwater medium in Hungate roll tubes with 1.5% melting agar and incubated at 55°C. After 2 months of incubation, fluorescent colonies (ca. 1 mm in diameter) were picked into saltwater medium in roll tubes with YE (0.5 g/liter), sodium acetate (0.1 mmol/liter), and trimethylamine (0.1 mmol/liter) under a gas mixture of H₂ and CO₂ (4/1, vol/vol, 200 kPa). After 1 month of incubation at 55°C, strain Tm2 was obtained. It was considered pure by the following criteria: no growth in purity test medium, no PCR amplification of bacterial 16S rRNA genes, and homogeneous sequences of archaeal 16S rRNA genes. Another methanogenic enrichment incubation at 60°C from oil production water was transferred repeatedly into saltwater medium with added YE and sodium acetate under a gas mixture of H₂ and CO₂ (4/1, vol/vol, 200 kPa). A hydrogenotrophic methanogen named HMD was purified by the serial-dilution method in liquid medium. Analysis of the 16S rRNA gene revealed that gene sequence of strain HMD was almost identical to that of strain Tm2^T (99.4% similarity). Therefore, strain Tm2^T was characterized further in detail.

Fig. 1.

Methane production and acetate degradation during the second transfer methanogenic enrichment. d, days.

Morphology.

Strain Tm2^T was slightly curved rods (0.3 μm in width and 2.2 to 5.9 μm in length) that occurred singly or in pairs. A flagellum was not observed, but some cells displayed a coccoid shape connected at the end of the rod cells (Fig. 2 A and B). Fluorescence microscopy indicated that these coccal cells contained F420 coenzyme (see Fig. S1 and S2 in the supplemental material), and 4′,6-diamidino-2-phenylindole (DAPI) staining suggested that they contained DNA (see Fig. S3 in the supplemental material). These coccoid-shaped cells were observed at both the log phase and the stationary phase. It remains unclear why the methanogen Tm2^T possesses two morphologies. White and irregular colonies (0.5 to 1 mm in diameter) were detected under a phase-contrast and immunofluorescence microscope. The cells of strain Tm2^T were not motile and were not lysed in 1% SDS.

Fig. 2.

Cellular morphology of strain Tm2^T. (A) Scanning electron micrograph. Bar, 2 μm. Arrows indicate coccoid-shaped cells. (B) Negative-stained micrograph. Bar, 5 μm.

Biochemical and physiological characterization.

Strain Tm2^T grew with H₂-CO₂ but not sodium formate, sodium acetate, trimethylamine, monomethylamine, ethanol, dimethyl sulfide, isopropanol, isobutanol, or 2-butanol. Acetate was a required growth factor in basal medium, while YE, coM, vitamin solutions, trace element solutions, selenite-tungstate solution, and NiCl₂ were not necessary for growth. Strain Tm2^T grew optimally at a pH of 6.9, no growth was observed at pH 6.6 or 8.9 (Fig. 3 A). The strain grew between 55 and 80°C, with the fastest growth at 65°C, but no growth occurred below 45°C or above 85°C (Fig. 3B). Strain Tm2^T grew fastest at an NaCl concentration of 0.025 g/liter and grew within a range of 0.025 to 30 g/liter. Minor growth was also detected above 40 g/liter NaCl (Fig. 3C). Based on its CH₄ production rate (44), the growth of strain Tm2^T was completely inhibited by chloramphenicol and rifampin, partially inhibited by ampicillin, apramycin, kanamycin, and neomycin, and not affected by penicillin G (see Fig. S1 in the supplemental material).

Fig. 3.

Influence of pH (A), temperature (B), and NaCl (C) on the growth of strain Tm2^T in 20 ml basal medium with sodium acetate, and/or YE in the headspace of H₂-CO₂.

Genomic and phylogenetic characterization.

The genomic DNA G+C content of strain Tm2^T was 40.1 ± 1.3 (mean ± standard deviation) or 41.1 mol%, based on the T_m or HPLC method, respectively. Analysis of the 16S rRNA gene revealed that the sequences of strain Tm2^T possessed 96.4% similarity to that of Methanothermobacter thermautotrophicus ΔH^T and Methanothermobacter wolfeii VKM B-1829^T (Fig. 4). The inferred amino acid sequence of the partial mcrA gene of strain Tm2^T showed 90.0% similarity to that of Methanobacterium ivanovii DSM 2611^T, Methanobacterium bryantii DSM 863^T, and Methanobacterium aarhusense H2-LR^T (Fig. 5) but had low bootstrap support (<50%) in phylogenetic treeing. The family Methanobacteriaceae currently comprises four genera, Methanobrevibacter, Methanosphaera, Methanobacterium, and Methanothermobacter. These genera have been isolated from various natural and artificial environments, such as the gastrointestinal tracts of animals, anaerobic bioreactors, rice paddy soils, and marine or freshwater sediments (3, 30, 37, 39, 45, 51). Most of the representatives of the family use H₂-CO₂ as a substrate for methanogenesis, except for Methanosphaera stadtmaniae, which depends on hydrogen and methanol (37, 39, 45). Thermophilic members in the family Methanobacteriaceae are assigned to the genus Methanothermobacter, which currently possesses three species (59). Methanothermobacter is the only genus of rod-shaped methanogens that grow on H₂-CO₂ at an optimum temperature of 60 to 65°C (3). Strain Tm2^T has similar characteristics, but in contrast to the type strains of the genus Methanothermobacter, Tm2^T requires acetate for growth (Table 1). In addition, the genomic DNA G+C content of strain Tm2^T was much lower than that of other members of the genus Methanothermobacter (Table 1). 16S rRNA gene analysis indicated that Tm2^T is related to the genus Methanothermobacter but shows a similarity of only 95.7 to 96.4% (Fig. 4), indicating that Tm2^T could not be assigned to the known species in the genus (14). Interestingly, the inferred amino acid sequence of the partial sequence of the mcrA gene from strain Tm2^T was most similar to that of the type strains of genus Methanobacterium (90%) but only distantly related to that of the species of the genus Methanothermobacter or Methanobacterium.

Fig. 4.

Phylogenetic tree based on 16S rRNA gene sequences of strain Tm2^T and related type species of the family Methanobacteriaceae using the neighbor-joining method by the MEGA 4.0 software based on 1,125 unambiguous bases and 1,000 bootstrap replications. The sequence of Methermicoccus shengliensis ZC-1^T (DQ787474) was used as the outgroup. Scale bar, 2% estimated difference in nucleotide sequence.

Fig. 5.

Phylogenetic tree of inferred amino acid sequences (159 amino acids) of the partial mcrA gene sequence of strain Tm2^T and related type strains in the family Methanobacteriaceae. The tree was constructed by using the neighbor-joining method in the MEGA 4.0 software with 1,000 bootstrap replications. The sequence of Methanosphaera stadtmaniae DSM 3091^T (AJ584650) was used as the outgroup. Bar, 5% estimated difference in amino acid sequence.

Table 1.

Characteristics of strain Tm2^T and type strains of genus Methanothermobacter

Strain	Cell width × length (μm)	Filaments	Gram staining	Use of H2-CO2 as catabolic substrate	Formate utilization	Chemoautotrophy	Acetate requirement	Growth-stimulating compound(s)a	pH range (optimum)	Temp (oC) range (optimum)	% Salinity (optimum)	Mol% G+C content (measurement method)	Source	Reference(s)
Tm2T	0.3 × 2.2-5.9	−	+	+	−	−	+		6.9-8.0 (6.9)	45-80 (65)	0-4 (0.5)	40.1 ± 1.3 (Tm), 41.1 (HPLC)	Oil sands	This study
M. defluvii ADZT	0.42 × 3-6	−	+	+	+	+	−	HS-coM, Ni2+, YE, Casamino Acids, sewage sludge	5.5-8.5 (6.5-7)	45-65 (60-65)	0-3 (0.08-2)	62.2 (Tm)	Anaerobic sludge	22
M. thermoflexus IDZT	0.4 × 7-20	−	+	+	+	+	−	HS-coM, Ni2+	6.0-9 (7.9-8.2)	45-70 (55)	0-5 (0.1-3)	55 (Tm)	Anaerobic sludge	22
M. thermophilus MT	0.36 × 1.4-6.5	+	−	+	−	+		HS-coM	7.0-8.5 (7.5)	45-65 (57)		44.7 (Tm)	Thermophilic anaerobic sewage sludge digester	4, 58
M. thermautotrophicus ΔHT	0.35-0.6 × 3-7	+	+	+	−	+	−		6-8 (7.2-7.6)	40-75 (65-70)		52 (Tm)	Sewage sludge	64
M. marburgensis MarburgT	0.4-0.6 × 3-6	+	+	+	−	+	−		5.0-8.0 (6.8-7.4)	45-70 (65)		47.6 (Tm)	Sewage sludge digester	4, 58
M. wolfeii VKM B-1829T	0.35-0.4 × 2.4-2.7	−	+	+	−	−	−	ND	6.0-8.2 (7.0-7.5)	37-76 (55-65)	0-2 (1)	61 ± 1 (Tm)	Mixture of sewage sludge and river sediment	62

^aHS-coM, coenzyme M; ND, not determined.

According to the minimal standards for describing new taxa of methanogens (2) and based on physiological and phylogenetic characteristics, we propose that strains Tm2^T and HMD represent a novel species of the genus Methanothermobacter and propose the name Methanothermobacter crinale sp. nov.

Ecological niche of the M. crinale-related phylotype in oil fields.

The 16S rRNA gene sequences with 98% (or greater) similarity to that of Tm2^T in published papers were thought to be members of the species M. crinale (see Table S1 in the supplemental material). A number of as-yet-uncultivated M. crinale-related phylotypes have been described in geographically distant high-temperature oil reservoirs such as oil fields on the north slope of Alaska (11, 15), the Yabase oil field in Japan (33), the Dagang oil field (40) and the Qinghuang unit in the People's Republic of China (26), high-temperature natural gas fields (38), and oil-polluted saline soil (63) (see Table S1 in the supplemental material). More interestingly, the occurrence of the M. crinale-related phylotype was found to be concomitant with syntrophic acetate oxidation. Using isotopic and molecular biological approaches, Mayumi et al. (33). demonstrated that syntrophic acetate oxidation coupled to hydrogenotrophic methanogenesis occurred in a high-temperature petroleum reservoir. The Thermacetogenium-related bacteria were responsible for syntrophic acetate oxidation, and prominent archaeal clone sequences (type clones YAB2A11 [AB539931] and YAB3A23 [AB539929]) were closely affiliated with that of M. crinale strain Tm2^T (99.2% and 98.8% sequence similarity, respectively). Five thermodynamically possible pathways for the conversion of hydrocarbons into methane have been proposed (10), and the incomplete oxidation of hydrocarbons, coupled with syntrophic acetate oxidation and hydrogenotrophic methanogenesis, is thought to be a key process in oil reservoirs (20). Gieg et al. (15). cultivated a thermophilic crude-oil-degrading methanogenic consortium from the production water of oil reservoirs and deduced that the predominant M. crinale-related phylotype (99.8% sequence similarity; GU357468) was likely to be involved in the syntrophic decomposition of crude oil. Nazina et al. (40) found that a Methanobacteriales-related consortium obtained from the Dagang oil field utilized ¹⁴CH₃-COONa and NaH¹⁴CO₃ to produce methane and suggested the presence of syntrophic acetate degradation via hydrogenotrophic methanogens. In that study, 45 clone sequences (type clone A1m_OTU3 [DQ097668]) out of 101 methanogenic archaeal clones belonged to the M. crinale phylotype (99.9% sequence similarity). In addition, this phylotype has also been detected in various thermophilic anaerobic reactors treating glucose (55) and mixtures of acetate, propionate, and sucrose (50), propionate and/or acetate (56), acetate and butyrate (57), and swine manure, sewage sludge, and a hot-rot compost suspension (23).

We found that methane production in the enrichment (before the isolation of Tm2^T) corresponded to acetate degradation at 55°C, while the aceticlastic methanogens were absent. Though without direct evidence, it was very likely that syntrophic acetate oxidation coupled to hydrogenotrophic methanogenesis occurred in this enrichment.

Description of Methanothermobacter crinale sp. nov.

Methanothermobacter crinale (cri.na′le. L. neut. n. crinale), a hairpin, referring to a special morphological feature of the genus Methanothermobacter, i.e., that some coccoid-shaped cells are attached to the end of the rod-shaped methanogen.

Cells occur singly or in pairs as straight or slightly curved rods (0.3 μm in width and 2.2 to 5.9 μm in length) but occasionally connected to coccoid-shaped cells at the ends. The cells are not motile, are not lysed in 1% SDS (wt/vol), and stain Gram positively. Methane is produced from H₂-CO₂, and acetate is a required growth factor. Good growth occurs at a pH of 6.9 (range, 6.6 to 8.9), a temperature of 65°C (range, 45 to 85°C), and an NaCl concentration of 0.025 g/liter (range, 0.025 to 40 g/liter). The genomic DNA G+C content is 40.1 ± 1.3 or 41.1 mol%, based on the T_m or HPLC method, respectively.

The type strain of Tm2^T (ACCC 00699^T = JCM 17393^T) was isolated from oil sands in the Haoxian central facility of in the Shengli oil field of the People's Republic of China.