Methanogenic paraffin degradation proceeds via alkane addition to fumarate by 'Smithella' spp. mediated by a syntrophic coupling with hydrogenotrophic methanogens.

Anaerobic microbial biodegradation of recalcitrant, water-insoluble substrates, such as paraffins, presents unique metabolic challenges. To elucidate this process, a methanogenic consortium capable of mineralizing long-chain n-paraffins (C28 -C50 ) was enriched from San Diego Bay sediment. Analysis of 16S rRNA genes indicated the dominance of Syntrophobacterales (43%) and Methanomicrobiales (26%). Metagenomic sequencing allowed draft genome assembly of dominant uncultivated community members belonging to the bacterial genus Smithella and the archaeal genera Methanoculleus and Methanosaeta. Five contigs encoding homologs of the catalytic subunit of alkylsuccinate synthase (assA) were detected. Additionally, mRNA transcripts for these genes, including a homolog binned within the 'Smithella' sp. SDB genome scaffold, were detected via RT-PCR, implying that paraffins are activated via 'fumarate addition'. Metabolic reconstruction and comparison with genome scaffolds of uncultivated n-alkane degrading 'Smithella' spp. are consistent with the hypothesis that syntrophically growing 'Smithella' spp. may achieve reverse electron transfer by coupling the reoxidation of ETFred to a membrane-bound FeS oxidoreductase functioning as an ETF:menaquinone oxidoreductase. Subsequent electron transfer could proceed via a periplasmic formate dehydrogenase and/or hydrogenase, allowing energetic coupling to hydrogenotrophic methanogens such as Methanoculleus. Ultimately, these data provide fundamental insight into the energy conservation mechanisms that dictate interspecies interactions salient to methanogenic alkane mineralization.

Dethiosulfatarculus sandiegensis gen. nov., sp. nov., isolated from a methanogenic paraffin-degrading enrichment culture and emended description of the family Desulfarculaceae.

A mesophilic deltaproteobacterium, designated strain SPRT, was isolated from a methanogenic consortium capable of degrading long-chain paraffins. Cells were motile, vibrio-shaped, and occurred singly, in pairs or in clusters. Strain SPRT did not metabolize hydrocarbons but grew fermentatively on pyruvate and oxaloacetate and autotrophically with H2 and CO2. Thiosulfate served as a terminal electron acceptor, but sulfate or sulfite did not. The organism required at least 10 g NaCl l- 1 and a small amount of yeast extract (0.001%) for growth. Optimal growth was observed between 30 and 37 °C and a pH range from 6.0 to 7.2. The DNA G+C content of SPRT's genome was 52.02 mol%. Based on 16S rRNA gene sequence analysis, strain SPRT was distinct from previously described Deltaproteobacteria, exhibiting the closest affiliation to Desulfarculus baarsii DSM 2075T and Desulfocarbo indianensis SCBMT, with only 91% similarity between their respective 16S gene sequences. In silico genome comparison supported the distinctiveness between strain SPRT and both Desulfocarbo indianensis SCBMT and Desulfarculus baarsii DSM 2075T. Based on physiological differences, as well as phylogenetic and genomic comparisons, we propose to classify SPRT as the type strain ( = DSM 100305T = JCM 30857T) of a novel species of a new genus with the name Dethiosulfatarculus sandiegensis gen. nov., sp. nov.

Youngiibacter fragilis gen. nov., sp. nov., isolated from natural gas production-water and reclassification of Acetivibrio multivorans as Youngiibacter multivorans comb. nov.

A taxonomic study employing a polyphasic approach was performed on a novel anaerobic bacterium isolated from natural gas production-water. The bacterium stained Gram-negative and consisted of non-motile, non-spore-forming, rod-shaped cells. Products of glucose or starch fermentation were ethanol, CO2, formate, acetate and H2. The predominant fatty acids were C16 : 0 ALDE and summed feature 3 comprising C16 : 1ω7c and/or C16 : 1ω6c. The DNA G+C content was 45.5 mol%. 16S rRNA gene sequence analysis demonstrated that the nearest phylogenetic neighbours of the novel strain were Acetivibrio multivorans DSM 6139(T) (98.5 %) and Proteiniclasticum ruminis JCM 14817(T) (95.4 %). The DNA-DNA hybridization value between the novel organism and Acetivibrio multivorans PeC1 DSM 6139(T) was determined to be only 30.2 %, demonstrating the separateness of the two species. Based on phylogenetic, phenotypic and chemotaxonomic evidence that clearly distinguished strain 232.1(T) from Proteiniclasticum ruminis and other close relatives, it is proposed that the novel isolate be classified as representing a novel species of a new genus within the family Clostridiaceae, Youngiibacter fragilis gen. nov., sp. nov. The type strain of the type species is 232.1(T) ( = ATCC BAA-2257(T) = DSM 24749(T)). In addition, Acetivibrio multivorans is proposed to be reclassified as Youngiibacter multivorans comb. nov.

The Complete Genome Sequence of n-Alkane-Degrading Desulfoglaeba alkanexedens ALDC Reveals Multiple Alkylsuccinate Synthase Gene Clusters.

Anaerobic alkane metabolism is critical in multiple environmental and industrial sectors, including environmental remediation, energy production, refined fuel stability, and biocorrosion. Here, we report the complete gap-closed genome sequence for a model n-alkane-degrading anaerobe, Desulfoglaeba alkanexedens ALDC.

Metabolic Capability of a Predominant Halanaerobium sp. in Hydraulically Fractured Gas Wells and Its Implication in Pipeline Corrosion.

Microbial activity associated with produced water from hydraulic fracturing operations can lead to gas souring and corrosion of carbon-steel equipment. We examined the microbial ecology of produced water and the prospective role of the prevalent microorganisms in corrosion in a gas production field in the Barnett Shale. The microbial community was mainly composed of halophilic, sulfidogenic bacteria within the order Halanaerobiales, which reflected the geochemical conditions of highly saline water containing sulfur species (S2O3 (2-), SO4 (2-), and HS(-)). A predominant, halophilic bacterium (strain DL-01) was subsequently isolated and identified as belonging to the genus Halanaerobium. The isolate could degrade guar gum, a polysaccharide polymer used in fracture fluids, to produce acetate and sulfide in a 10% NaCl medium at 37°C when thiosulfate was available. To mitigate potential deleterious effects of sulfide and acetate, a quaternary ammonium compound was found to be an efficient biocide in inhibiting the growth and metabolic activity of strain DL-01 relative to glutaraldehyde and tetrakis (hydroxymethyl) phosphonium sulfate. Collectively, our findings suggest that predominant halophiles associated with unconventional shale gas extraction could proliferate and produce sulfide and acetate from the metabolism of polysaccharides used in hydraulic fracturing fluids. These metabolic products might be returned to the surface and transported in pipelines to cause pitting corrosion in downstream infrastructure.

Field and laboratory studies on the bioconversion of coal to methane in the San Juan Basin.

The bioconversion of coal to methane in the San Juan Basin, New Mexico, was investigated. Production waters were analyzed via enrichment studies, metabolite-profiling, and culture-independent methods. Analysis of 16S rRNA gene sequences indicated the presence of methanogens potentially capable of acetoclastic, hydrogenotrophic, and methylotrophic metabolisms, predominantly belonging to the Methanosarcinales and Methanomicrobiales. Incubations of produced water and coal readily produced methane, but there was no correlation between the thermal maturity and methanogenesis. Coal methanogenesis was greater when samples with a greater richness of Firmicutes were utilized. A greater archaeal diversity was observed in the presence of several aromatic and short-chain fatty acid metabolites. Incubations amended with lactate, hydrogen, formate, and short-chain alcohols produced methane above un-amended controls. Methanogenesis from acetate was not observed. Metabolite profiling showed the widespread occurrence of putative aromatic ring intermediates including benzoate, toluic acids, phthalic acids, and cresols. The detection of saturated and unsaturated alkylsuccinic acids indicated n-alkane and cyclic alkane/alkene metabolism. Microarray analysis complemented observations based on hybridization to functional genes related to the anaerobic metabolism of aromatic and aliphatic substrates. These data suggest that coal methanogenesis is unlikely to be limited by methanogen biomass, but rather the activation and degradation of coal constituents.