Lirong Dai

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, Tm2T and HMD, from the oil sands and oil production water in the Shengli oil field in the Peoples Republic of China. The type strain, Tm2T, 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 Tm2T grew with H2-CO2, but acetate is required. Optimum growth of strain Tm2T 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 Tm2T was most closely related to Methanothermobacter thermautotrophicus ΔHT and Methanothermobacter wolfeii VKM B-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 (ACCC 00699T = JCM 17393T).

DNA-SIP Reveals That Syntrophaceae Play an Important Role in Methanogenic Hexadecane Degradation

The methanogenic degradation of linear alkanes is a common process in oil-impacted environments. However, little is known about the key players involved in this process. Here, the hexadecane-degrading organisms in a methanogenic, hexadecane-degrading consortium designated M82 obtained from Shengli oilfield and maintained at 35°C for over 4 years, were identified by DNA-stable isotope probing with UL-13C-hexadecane, followed by density-resolved terminal restriction fragment length polymorphism (T-RFLP) analysis, cloning and phylogenetic analysis of 16S rRNA gene fragments. Compared to the fractions of the 12C treatment, the relative abundance of two phylotypes significantly increased in the heavy fractions of the 13C-hexadecane incubated microcosm. One belongs to a uncultured member of the bacterial family Syntrophaceae, which show 95–97% rRNA sequence identity with Smithella propionica, and the other is affiliated with Methanoculleus receptaculi (>99% sequence identity). The results of the present study prove the significant role of uncultured Syntrophaceae in degradation of hexadecane, probably through syntrophic interactions with hydrogenotrophic methanogens.

Novel bacterial groups dominate in a thermophilic methanogenic hexadecane‐degrading consortium

A methanogenic hexadecane-degrading consortium designated SK originating from the Shengli oil field was cultured at 55 °C. The structure and dynamics of the microbial community during successive transfers were examined using terminal restriction fragment length polymorphism (T-RFLP) fingerprinting and sequencing of 16S rRNA gene fragments. The archaeal community was mainly composed of hydrogenotrophic methanogens affiliated with Methanothermobacter crinale and acetoclastic methanogens related to Methanosaeta thermophila. Over four-fifths of the bacterial clones in the hexadecane-degrading subcultures exhibited < 90% similarity to sequences of known type strains, and clones were mainly grouped into unclassified bacteria (66.3-66.7%), Firmicutes (9.6-10.6%), Thermotogae (7.0-7.7%), and Nitrospira (5.3-5.8%). The dominant operating taxonomic unit (OTU) (41.3-43.0% of all clones) representing terminal restriction fragment (T-RF) 125 bp exhibited only 82.6% sequence similarity to Thermotoga maritime and clustered in a monophyletic, deep-branching lineage (designated Shengli cluster). Two other OTUs (T-RFs 66 and 67 bp) were assigned to uncultured members of the candidate phylum OP8 and Firmicutes, respectively. These novel bacterial assemblages are likely to be involved in the process of hexadecane degradation because of their high abundance in the enrichments. These result substantially expand the knowledge of the extent of bacterial diversity associated with the anaerobic degradation of alkanes under thermophilic conditions.

Petroclostridium xylanilyticum gen. nov., sp. nov., a xylan-degrading bacterium isolated from an oilfield, and reclassification of clostridial cluster III members into four novel genera in a new Hungateiclostridiaceae fam. nov.

A rod-shaped, Gram-stain-positive, obligately anaerobic, xylan-degrading bacterium, SK-Y3T, was isolated from oily-sludge of Shengli oilfield, China. Optimum growth occurred at 50 °C, at pH 7.5 and without addition of NaCl. The predominant cellular fatty acids of strain SK-Y3T were iso-C15 : 0, anteiso-C15 : 0 and iso-C17 : 0, and the main polar lipids were glycolipids (GL), lipids (L), phosphatidylglycerol (PG) and diphosphatidylglycerol (DPG); no respiratory quinones were detected. The genomic DNA G+C content was 37.2 mol%. Phylogenetic analysis of 16S rRNA gene sequences showed that strain SK-Y3T belongs to clostridial cluster III, exhibiting 91-92% sequence similarity to the most closely related species, namely Clostridium clariflavum, Clostridium straminisolvens and Acetivibrio cellulolyticus. Based on distinct physiological and phylogenetic differences from the aforementioned described taxa, strain SK-Y3T (=DSM 103557T=ACCC 19952T) is proposed as the type strain of a novel species of a new genus, Petroclostridium xylanilyticum gen. nov., sp. nov. Furthermore, analysis through 16S rRNA gene, ribosomal protein and whole genome sequences indicated that clostridial cluster III members should be reclassified into four novel genera for which the names Hungateiclostridium gen. nov., Thermoclostridium gen. nov., Ruminiclostridium gen. nov. and Pseudoclostridium gen. nov. are proposed. In combination with the genera Anaerobacterium, Cellulosibacter, Ercella, Fastidiosipila, Mageeibacillus, Pseudobacteroides, Petroclostridium and Saccharofermentans, clostridial cluster III members formed a monophyletic clade within the order Clostridiales but that was clearly distinguished from other Ruminococcaceae members, which is proposed as a novel family, Hungateiclostridiaceae fam. nov.