isolation and characterization of methanothermobacter ... · ization of two strains, tm2t and hmd,...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 2011, p. 5212–5219 Vol. 77, No. 150099-2240/11/$12.00 doi:10.1128/AEM.00210-11Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Isolation and Characterization of Methanothermobacter crinalesp. nov., a Novel Hydrogenotrophic Methanogen from

the Shengli Oil Field�†Lei Cheng,1,2 Lirong Dai,2 Xia Li,2 Hui Zhang,2 and Yahai Lu1*

College of Resources and Environmental Sciences, China Agricultural University, Beijing 100193, China,1 andKey Laboratory of Energy Microbiology and Its Application of Ministry of Agriculture,

Biogas Institute of Ministry of Agriculture, Chengdu 610041, China2

Received 30 January 2011/Accepted 9 June 2011

Syntrophic acetate oxidation coupled with hydrogenotrophic methanogenesis is an alternative methanogenicpathway in certain thermophilic anaerobic environments such as high-temperature oil reservoirs and ther-mophilic biogas reactors. In these environments, the dominant thermophilic methanogens were generallyrelated to uncultured organisms of the genus Methanothermobacter. Here we isolated two representative strains,Tm2T and HMD, from the oil sands and oil production water in the Shengli oil field in the People’s Republicof China. The type strain, Tm2T, was nonmotile and stained Gram positive. The cells were straight to slightlycurved rods (0.3 �m in width and 2.2 to 5.9 �m in length), but some of them possessed a coccal shapeconnecting with the rods at the ends. Strain Tm2T grew with H2-CO2, but acetate is required. Optimum growthof strain Tm2T occurred in the presence of 0.025 g/liter NaCl at pH 6.9 and a temperature of 65°C. The G�Ccontent 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 Tm2T

was most closely related to Methanothermobacter thermautotrophicus �HT and Methanothermobacter wolfeii VKMB-1829T (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 Tm2T (ACCC00699T � JCM 17393T).

Methanogenesis is the terminal process of organic com-pound degradation and plays a major role in the global carboncycle, occurring in a variety of natural and artificial environ-ments, such as the gastrointestinal tracts of animals, rice paddysoils, deep subsurface marine or freshwater sediments, andanaerobic bioreactors (14, 28). The most important precursorsfor methane production during anaerobic digestion of organicmatter are acetate and H2-CO2, which are converted intomethane by aceticlastic and hydrogenotrophic methanogens(28), respectively. However, an alternative pathway for acetateis syntrophic acetate oxidation followed by hydrogenotrophicmethanogenesis (65). Several bacteria responsible for acetateoxidation have been isolated and characterized (17, 25, 48, 60),and a series of reports revealed that this process is present inhigh-temperature oil reservoirs (15, 33, 40), thermophilic ormesophilic biogas reactors (16, 21, 23, 43, 49), lake sediments(41), and rice paddy soils (27). Interestingly, it is proposed thatsyntrophic acetate oxidation coupled to hydrogenotrophicmethanogenesis is the main methanogenic pathway in high-temperature petroleum reservoirs (33) and probably involvedin the thermophilic methanogenic degradation of hydrocarbon(15). Cloning of rRNA genes from these environments indi-

cates that thedominant thermophilichydrogenotrophicmethan-ogens were found to be affiliated with the genus Methanother-mobacter. However, to our knowledge, pure isolates have notbeen described. Here, we report the isolation and character-ization of two strains, Tm2T and HMD, belonging to a novelphylotype of Methanothermobacter.

MATERIALS AND METHODS

Samples and media. The production water of oil reservoirs was collected fromthe 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 apressure of 9.24 MPa. The in situ temperature of the reservoir ranges from 75°Cto 80°C, and the total salt concentration of the oil production water is 9,794mg/liter. The oil sands comprised crude oil (30.2%), water (52.8%), and othersolid sands. They were sampled from oil-water separating tanks at the Haoxiancentral facility of the Shengli oil field in 2003 and maintained at room temper-ature.

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-cysteinehydrochloride (13) was used for enrichment and purification. It was autoclavedfor 30 min at 121°C and then reduced with Na2S � 9H2O (0.03%). A solution ofsterile NaHCO3 (0.25%), a vitamin solution (2 ml/liter) (34), trace elementsolution SL-7 (2 ml/liter) (61), vitamin B12 (2 ml/liter) (13), and vitamin B1 (2ml/liter) (13) were injected into the medium before inoculation, and the pH wasadjusted to 7.0 to 7.2. The basal medium used was composed of 0.5 g NaCl, 0.5 gMgCl � 6H2O, 0.1 g CaCl2 � 2H2O, 0.3 g NH4Cl, 0.2 g KH2PO4, 0.5 g KCl, and0.5 g L-cysteine hydrochloride (13). Modified DSM medium 119 was preparedwith (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 (preparedaccording to the instructions for DSM medium 119), 2.5 g NaHCO3, 0.5 gcysteine-HCl � H2O, and 0.5 g Na2S � 9H2O, and the pH was adjusted to 6.7 to7.0. Aliquots were distributed into glass vials sealed with butyl rubber stoppersand aluminum caps or transferred into anaerobic tubes. All of the media were

* Corresponding author. Mailing address: College of Resourcesand Environmental Sciences, China Agricultural University, Beijing100193, China. Phone and fax: 86-10-62733617. E-mail: [email protected].

† Supplemental material for this article may be found at http://aem.asm.org/.

� Published ahead of print on 24 June 2011.

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prepared anaerobically under a gas atmosphere of 80% N2 and 20% CO2 via theHungate anaerobic technique (31), in which resazurin (1 mg/liter) is a redoxindicator.

Enrichment and purification. Samples (about 10 g) of oil sands were distrib-uted into 120-ml vials containing 50 ml saltwater medium and incubated staticallyat 55°C in the dark. After 118 days, the initial preenrichment with positivemethane production was transferred into fresh saltwater medium and incubatedstatically at 55°C in the dark.

After several months of incubation, the Hungate roll tube and serial-dilutionmethods were applied for the isolation of pure cultures (18). Approximately0.5-ml methanogenic enrichments were serially diluted in 5 ml of saltwatermedium in roll tubes and incubated at 55°C with 1.5% melting agar amendedwith sodium acetate (0.1 mmol/liter), trimethylamine (0.1 mmol/liter), and a gasmixture 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), sodiumacetate (0.1 mmol/liter), and trimethylamine (0.1 mmol/liter) under a gas mixtureof H2 and CO2 (4/1, vol/vol, 200 kPa). After several repeated isolations, purecultures were obtained and assessed with a purity test medium that contained4.0 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). Puritytests were conducted under both anaerobic and aerobic conditions.

The other methanogenic enrichment was obtained from a 10-ml mixture of oilproduction water in 50 ml medium at 60°C, and a serial-dilution method wasused 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 conductivitydetector. The column, oven, and detector temperatures were 50°C, 50°C, and70°C, respectively. The carrier gas was hydrogen (99.999%) at a flow rate of 50ml/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 lawafter calibration with a gas mixture of N2, CH4, and CO2 at 29.96%, 39.99%, and30.05%, respectively.

Volatile fatty acids (VFA) were analyzed by gas chromatography (GC; Shi-madzu GC-7AG) with a stainless steel column packed with GDX103 (pretreat-ment with 5% phosphoric acid) and a flame ionization detector. The tempera-tures 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 ofnitrogen, hydrogen, and air were 70, 50, and 500 ml/min, respectively. Afteracidification with H2SO4 to pH �2 and centrifugation, 10 �l of supernatant wasinjected for GC analysis with a data processor (Shimadzu C-R1B). A mixture of100 ppm acetic acid, 100 ppm propionic acid, and100 ppm butyric acid was usedas an external standard.

Growth and genomic characteristics. (i) Microscopy. Cultures were regularlyobserved under a phase-contrast and immunofluorescence microscope (Nikon80i). The Gram reaction was determined as described previously (2). A Hitachi3400N scanning electron microscope was used to observe cell morphology (5);cells were negatively stained with 1% uranyl acetate and observed in a JEM 1230transmission electron microscope (36).

(ii) Biochemical and physiological analyses. To investigate potential sub-strates, sodium formate (20 mM), sodium acetate (20 mM), trimethylamine (20mM), 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 DSMmedium 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 asfollows: (i) a mixture of coenzyme M (coM; 25 mg/liter), YE (1 g/liter), andvitamins and trace element solution SL-7 (2 ml/liter) (34, 61); (ii) coM (25mg/liter); (iii) sludge fluid (10 ml/liter); (iv) a mixture of vitamin and traceelements solutions SL-7 (2 ml/liter) (34, 61); (v) YE (1 g/liter); (vi) sodiumacetate (10 mM); (vii) NiCl2 (0.01 g/liter); and (viii) selenite-tungstate solution(0.01 ml/liter) (52). The effect of pH on strain Tm2T was determined in basalmedium 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 pH7, 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 pH7.5 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 Tm2T was determinedin 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 Tm2T wasdetermined in basal medium amended with sodium acetate (0.1 g/liter) at a pHof 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 triplicatewith 20 ml of medium (60-ml vials) which was inoculated (5%, vol/vol) with aculture in the exponential growth phase.

(iii) Susceptibility experiments. The sensitivity of strain Tm2T to the antibi-otics 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 mediumamended 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 treat-ment after grinding under liquid N2 as previously described (19), and its G�Ccontent was determined by the thermal denaturation (Tm) method (6, 32) andthe high-performance liquid chromatography (HPLC) method (35). Thegenomic DNA of Escherichia coli K-12 was used as a reference.

Phylogenetic analysis. About 1.5 ml of liquid culture was collected for genomicDNA extraction by bead beating (42). DNA products were purified with thePromega Wizard DNA cleanup system (Promega) and stored at �25°C. The 16SrRNA and mcrA gene fragments were amplified using primers Ar21F/1492R,Ar109f/Ar915r, and MCRf/MCRr, respectively (8, 29, 53), reaction mixtures of50 �l consisted of 10 to 50 ng template DNA, 5 �l 10� PCR buffer, 4 �ldeoxynucleoside triphosphates (each at 2.5 mM), 3 �l MgCl2 (25 mM), 0.25 �lTaKaRa Taq (5 U/�l), and 1 �l each of the forward and reverse primers (10 �Mfor the 16S rRNA gene and 50 �M for the mcrA gene). Amplification witharchaeal 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, witha final extension at 72°C for 7 min. Amplification with archaeal primers Ar109fand Ar915r was described previously (42). PCR cycles of mcrA genes were asfollows: 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 gelrecovery kit (LangGang Biotech Co., Ltd.). The cloning and sequencing of 16SrRNA and partial mcrA gene fragments were conducted according to Rui et al.(46). The 16S rRNA gene sequences were checked for chimeras with the Bel-lerophon program of the Greengene database (9), and aligned with sequences inRibosomal Database Project release 10 to search for the most closely relatedsequences (1, 7). The 16S rRNA gene sequences of the methanogenic enrich-ment clone were aligned with the ClustalX software (24), the distance matriceswere 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 a98% threshold (47). Phylogenetic trees were constructed and compared forconsistency by using the neighbor-joining, minimum-evolution, and maximum-

FIG. 1. Methane production and acetate degradation during thesecond transfer methanogenic enrichment. d, days.

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parsimony methods in the MEGA 4.0 software (54). Bootstrap values werecalculated after 1,000 replications.

Nucleotide sequence accession numbers. The 16S rRNA gene sequences ofstrains Tm2T and HMD and the partial mcrA gene sequence of strain Tm2T weredeposited in the GenBank database with accession numbers HQ283273,HQ828065, and HQ283274, respectively. The GenBank accession numbers ofmethanogenic enrichment clones are HQ845184 to HQ845192.

RESULTS AND DISCUSSION

Enrichment and purification. The initial preenrichmentfrom oil sands with positive methane production was trans-ferred into fresh saltwater medium (80 ml). Methane produc-tion was steady for 87 days at an average rate of 2.5 �mol/daybefore decreasing to approximately 0.7 �mol/day. The mainVFA detected was acetate, which declined from 205 �mol atday 87 to 34 �mol after 302 days (Fig. 1), suggesting thatmethane was produced from acetate degradation (Fig. 1). Pro-pionate was also detected at a low concentration. A molecularsurvey of the archaeal 16S rRNA gene clone library con-structed from the first transfer of methanogenic enrichment at302 days revealed that the archaeal community consisted oftwo groups OTU1 (6 clones) and OTU2 (3 clones), which wereaffiliated with the type strains of Methanothermobacter wolfeiiDSM 2970T (95.7% sequence similarity, AB104858) andMethanolinea tarda NOBI-1T (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 incu-bation, fluorescent colonies (ca. 1 mm in diameter) werepicked into saltwater medium in roll tubes with YE (0.5 g/liter), sodium acetate (0.1 mmol/liter), and trimethylamine (0.1mmol/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 wasobtained. It was considered pure by the following criteria: nogrowth in purity test medium, no PCR amplification of bacte-rial 16S rRNA genes, and homogeneous sequences of archaeal16S rRNA genes. Another methanogenic enrichment incuba-tion at 60°C from oil production water was transferred repeat-edly into saltwater medium with added YE and sodium acetateunder a gas mixture of H2 and CO2 (4/1, vol/vol, 200 kPa). Ahydrogenotrophic methanogen named HMD was purified bythe serial-dilution method in liquid medium. Analysis of the16S rRNA gene revealed that gene sequence of strain HMD

FIG. 2. Cellular morphology of strain Tm2T. (A) Scanning electronmicrograph. Bar, 2 �m. Arrows indicate coccoid-shaped cells. (B) Neg-ative-stained micrograph. Bar, 5 �m.

FIG. 3. Influence of pH (A), temperature (B), and NaCl (C) on thegrowth of strain Tm2T in 20 ml basal medium with sodium acetate,and/or YE in the headspace of H2-CO2.

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was almost identical to that of strain Tm2T (99.4% similarity).Therefore, strain Tm2T was characterized further in detail.

Morphology. Strain Tm2T was slightly curved rods (0.3 �min width and 2.2 to 5.9 �m in length) that occurred singly or inpairs. A flagellum was not observed, but some cells displayed a

coccoid shape connected at the end of the rod cells (Fig. 2Aand B). Fluorescence microscopy indicated that these coccalcells contained F420 coenzyme (see Fig. S1 and S2 in thesupplemental material), and 4�,6-diamidino-2-phenylindole(DAPI) staining suggested that they contained DNA (see Fig.

FIG. 4. Phylogenetic tree based on 16S rRNA gene sequences of strain Tm2T and related type species of the family Methanobacteriaceae usingthe neighbor-joining method by the MEGA 4.0 software based on 1,125 unambiguous bases and 1,000 bootstrap replications. The sequence ofMethermicoccus shengliensis ZC-1T (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 Tm2T and relatedtype strains in the family Methanobacteriaceae. The tree was constructed by using the neighbor-joining method in the MEGA 4.0 software with1,000 bootstrap replications. The sequence of Methanosphaera stadtmaniae DSM 3091T (AJ584650) was used as the outgroup. Bar, 5% estimateddifference in amino acid sequence.



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S3 in the supplemental material). These coccoid-shaped cellswere observed at both the log phase and the stationary phase.It remains unclear why the methanogen Tm2T possesses twomorphologies. White and irregular colonies (0.5 to 1 mm indiameter) were detected under a phase-contrast and immuno-fluorescence microscope. The cells of strain Tm2T were notmotile and were not lysed in 1% SDS.

Biochemical and physiological characterization. StrainTm2T grew with H2-CO2 but not sodium formate, sodiumacetate, trimethylamine, monomethylamine, ethanol, dimethylsulfide, isopropanol, isobutanol, or 2-butanol. Acetate was arequired growth factor in basal medium, while YE, coM, vita-min solutions, trace element solutions, selenite-tungstate solu-tion, and NiCl2 were not necessary for growth. Strain Tm2T

grew optimally at a pH of 6.9, no growth was observed at pH6.6 or 8.9 (Fig. 3A). The strain grew between 55 and 80°C, withthe fastest growth at 65°C, but no growth occurred below 45°Cor above 85°C (Fig. 3B). Strain Tm2T grew fastest at an NaClconcentration of 0.025 g/liter and grew within a range of 0.025to 30 g/liter. Minor growth was also detected above 40 g/literNaCl (Fig. 3C). Based on its CH4 production rate (44), thegrowth of strain Tm2T was completely inhibited by chloram-phenicol and rifampin, partially inhibited by ampicillin, apra-mycin, kanamycin, and neomycin, and not affected by penicillinG (see Fig. S1 in the supplemental material).

Genomic and phylogenetic characterization. The genomicDNA G�C content of strain Tm2T was 40.1 � 1.3 (mean �standard deviation) or 41.1 mol%, based on the Tm or HPLCmethod, respectively. Analysis of the 16S rRNA gene revealedthat the sequences of strain Tm2T possessed 96.4% similarityto that of Methanothermobacter thermautotrophicus �HT andMethanothermobacter wolfeii VKM B-1829T (Fig. 4). The in-ferred amino acid sequence of the partial mcrA gene of strainTm2T showed 90.0% similarity to that of Methanobacteriumivanovii DSM 2611T, Methanobacterium bryantii DSM 863T,and Methanobacterium aarhusense H2-LRT (Fig. 5) but hadlow bootstrap support (�50%) in phylogenetic treeing. Thefamily Methanobacteriaceae currently comprises four genera,Methanobrevibacter, Methanosphaera, Methanobacterium, andMethanothermobacter. These genera have been isolated fromvarious natural and artificial environments, such as the gastro-

intestinal tracts of animals, anaerobic bioreactors, rice paddysoils, and marine or freshwater sediments (3, 30, 37, 39, 45, 51).Most of the representatives of the family use H2-CO2 as asubstrate for methanogenesis, except for Methanosphaerastadtmaniae, which depends on hydrogen and methanol (37,39, 45). Thermophilic members in the family Methanobacteri-aceae are assigned to the genus Methanothermobacter, whichcurrently possesses three species (59). Methanothermobacteris the only genus of rod-shaped methanogens that grow onH2-CO2 at an optimum temperature of 60 to 65°C (3). StrainTm2T has similar characteristics, but in contrast to the typestrains of the genus Methanothermobacter, Tm2T requiresacetate for growth (Table 1). In addition, the genomic DNAG�C content of strain Tm2T was much lower than that ofother members of the genus Methanothermobacter (Table 1).16S rRNA gene analysis indicated that Tm2T is related to thegenus Methanothermobacter but shows a similarity of only 95.7to 96.4% (Fig. 4), indicating that Tm2T could not be assignedto the known species in the genus (14). Interestingly, the in-ferred amino acid sequence of the partial sequence of themcrA gene from strain Tm2T was most similar to that of thetype strains of genus Methanobacterium (90%) but only dis-tantly related to that of the species of the genus Methanother-mobacter or Methanobacterium.

According to the minimal standards for describing new taxaof methanogens (2) and based on physiological and phyloge-netic characteristics, we propose that strains Tm2T and HMDrepresent a novel species of the genus Methanothermobacterand propose the name Methanothermobacter crinale sp. nov.

Ecological niche of the M. crinale-related phylotype in oilfields. The 16S rRNA gene sequences with 98% (or greater)similarity to that of Tm2T in published papers were thought tobe members of the species M. crinale (see Table S1 in thesupplemental material). A number of as-yet-uncultivated M.crinale-related phylotypes have been described in geographi-cally distant high-temperature oil reservoirs such as oil fieldson the north slope of Alaska (11, 15), the Yabase oil field inJapan (33), the Dagang oil field (40) and the Qinghuang unitin the People’s Republic of China (26), high-temperature nat-ural gas fields (38), and oil-polluted saline soil (63) (see TableS1 in the supplemental material). More interestingly, the oc-

TABLE 1. Characteristics of strain Tm2T and type strains of genus Methanothermobacter

Strain Cell width � length (�m) Filaments Gramstaining

Use of H2-CO2as catabolic

substrateFormate

utilization Chemoautotrophy Acetaterequirement

Tm2T 0.3 � 2.2–5.9 � � � � � �

M. defluvii ADZT 0.42 � 3–6 � � � � � �

M. thermoflexus IDZT 0.4 � 7–20 � � � � � �M. thermophilus MT 0.36 � 1.4–6.5 � � � � �

M. thermautotrophicus �HT 0.35–0.6 � 3–7 � � � � � �M. marburgensis MarburgT 0.4–0.6 � 3–6 � � � � � �M. wolfeii VKM B-1829T 0.35–0.4 � 2.4–2.7 � � � � � �

a HS-coM, coenzyme M; ND, not determined.



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currence of the M. crinale-related phylotype was found to beconcomitant with syntrophic acetate oxidation. Using isoto-pic and molecular biological approaches, Mayumi et al. (33).demonstrated that syntrophic acetate oxidation coupled tohydrogenotrophic methanogenesis occurred in a high-tem-perature petroleum reservoir. The Thermacetogenium-re-lated bacteria were responsible for syntrophic acetate oxi-dation, and prominent archaeal clone sequences (typeclones YAB2A11 [AB539931] and YAB3A23 [AB539929])were closely affiliated with that of M. crinale strain Tm2T

(99.2% and 98.8% sequence similarity, respectively). Fivethermodynamically possible pathways for the conversion ofhydrocarbons into methane have been proposed (10), and theincomplete oxidation of hydrocarbons, coupled with syntrophicacetate oxidation and hydrogenotrophic methanogenesis, isthought to be a key process in oil reservoirs (20). Gieg et al.(15). cultivated a thermophilic crude-oil-degrading methano-genic consortium from the production water of oil reservoirsand deduced that the predominant M. crinale-related phylo-type (99.8% sequence similarity; GU357468) was likely to beinvolved in the syntrophic decomposition of crude oil. Nazinaet al. (40) found that a Methanobacteriales-related consortiumobtained from the Dagang oil field utilized 14CH3-COONa andNaH14CO3 to produce methane and suggested the presence ofsyntrophic acetate degradation via hydrogenotrophic methano-gens. In that study, 45 clone sequences (type clone A1m_OTU3 [DQ097668]) out of 101 methanogenic archaeal clonesbelonged to the M. crinale phylotype (99.9% sequence similar-ity). In addition, this phylotype has also been detected in var-ious thermophilic anaerobic reactors treating glucose (55) andmixtures of acetate, propionate, and sucrose (50), propionateand/or acetate (56), acetate and butyrate (57), and swine ma-nure, sewage sludge, and a hot-rot compost suspension (23).

We found that methane production in the enrichment (be-fore the isolation of Tm2T) corresponded to acetate degrada-tion at 55°C, while the aceticlastic methanogens were absent.Though without direct evidence, it was very likely that syn-trophic acetate oxidation coupled to hydrogenotrophic methan-ogenesis occurred in this enrichment.

Description of Methanothermobacter crinale sp. nov. Methano-thermobacter 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 areattached to the end of the rod-shaped methanogen.

Cells occur singly or in pairs as straight or slightly curvedrods (0.3 �m in width and 2.2 to 5.9 �m in length) but occa-sionally connected to coccoid-shaped cells at the ends. Thecells are not motile, are not lysed in 1% SDS (wt/vol), and stainGram positively. Methane is produced from H2-CO2, andacetate is a required growth factor. Good growth occurs at apH of 6.9 (range, 6.6 to 8.9), a temperature of 65°C (range, 45to 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 Tm or HPLC method, respec-tively.

The type strain of Tm2T (ACCC 00699T JCM 17393T) wasisolated from oil sands in the Haoxian central facility of in theShengli oil field of the People’s Republic of China.

ACKNOWLEDGMENTS

We thank Xiaoxia Zhang for assistance in determining GC mol%contents, Zhe Lv for supplying antibiotics, and Qiang Li and SichunMa for the analysis of VFA. We appreciate William B. Whitman forhelpful suggestions and comments on the manuscript and the taxon-omy of Tm2. We also thank Jean Euzeby for the etymology of thenovel taxon.

This study was supported partly by National Natural Science Foun-dation of China (grants 3090049, 40625003, and 4097059) and theChang Jiang Scholars Program of the Chinese Ministry of Education.

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TABLE 1—Continued

Growth-stimulatingcompound(s)a pH range (optimum) Temp (oC)

range (optimum)% Salinity(optimum)

Mol% G�Ccontent

(measurementmethod)

Source Reference(s)

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

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

HS-coM, Ni2� 6.0–9 (7.9–8.2) 45–70 (55) 0–5 (0.1–3) 55 (Tm) Anaerobic sludge 22HS-coM 7.0–8.5 (7.5) 45–65 (57) 44.7 (Tm) Thermophilic anaerobic sewage

sludge digester4, 58

6–8 (7.2–7.6) 40–75 (65–70) 52 (Tm) Sewage sludge 645.0–8.0 (6.8–7.4) 45–70 (65) 47.6 (Tm) Sewage sludge digester 4, 58

ND 6.0–8.2 (7.0–7.5) 37–76 (55–65) 0–2 (1) 61 � 1 (Tm) Mixture of sewage sludge andriver sediment

62



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