Microbial communities and their roles in the Cenozoic sulfurous oil reservoirs in the Southwestern Qaidam Basin, Western China.

The latest discovery of sulfurous natural gas marked a breakthrough in the Cenozoic natural gas exploration in the southwestern margin of Qaidam Basin. The 16S rRNA analyses were performed on the crude oil samples from H2S-rich reservoirs in the Yuejin, Shizigou and Huatugou profiles, to understand the sulfurous gas origin, which was also integrated with carbon and hydrogen isotopes of alkane and sulfur isotopes of H2S collected from the Yingxiongling Area. Results show that the microorganisms in samples can survive in the hypersaline reservoirs, and can be classified into multiple phyla, including Proteobacteria, Planctomycetes, Firmicutes, Bacteroidetes, and Haloanaerobiaeota. Methanogens are abundant in all of the three profiles, while sulfate-reducing bacteria are abundant in Yuejin and Huatugou profiles, contributing to the methane and H2S components in the natural gas. The carbon, hydrogen and sulfur isotopes of sulfurous natural gas in the Yingxiongling Area show that the natural gas is a mixture of coal-type gas and oil-type gas, which was primarily derived from thermal degradation, and natural gas from the Yuejin and Huatugou profiles also originated from biodegradation. The isotopic analysis agrees well with the 16S rRNA results, i.e., H2S-rich natural gas from the Cenozoic reservoirs in the southwest margin of the Qaidam Basin was primarily of thermal genesis, with microbial genesis of secondary importance.

The effect of lanthanum on growth and gene expression in a facultative methanotroph.

The biological importance of lanthanides has only recently been identified, initially as the active site metal of the alternative methanol dehydrogenase (MDH) Xox-MDH. So far, the effect of lanthanide (Ln) has only been studied in relatively few organisms. This work investigated the effects of Ln on gene transcription and protein expression in the facultative methanotroph Methylocella silvestris BL2, a widely distributed methane-oxidizing bacterium with the unique ability to grow not just on methane but also on other typical components of natural gas, ethane and propane. Expression of calcium- or Ln-dependent MDH was controlled by Ln (the lanthanide switch) during growth on one-, two- or three-carbon substrates, and Ln imparted a considerable advantage during growth on propane, a novel result extending the importance of Ln to consumers of this component of natural gas. Two Xox-MDHs were expressed and regulated by Ln in M. silvestris, but interestingly Ln repressed rather than induced expression of the second Xox-MDH. Despite the metabolic versatility of M. silvestris, no other alcohol dehydrogenases were expressed, and in double-mutant strains lacking genes encoding both Ca- and Ln-dependent MDHs (mxaF and xoxF5 or xoxF1), growth on methanol and ethanol appeared to be enabled by expression of the soluble methane monooxygenase.

Synthetic Biology on Acetogenic Bacteria for Highly Efficient Conversion of C1 Gases to Biochemicals.

Synthesis gas, which is mainly produced from fossil fuels or biomass gasification, consists of C1 gases such as carbon monoxide, carbon dioxide, and methane as well as hydrogen. Acetogenic bacteria (acetogens) have emerged as an alternative solution to recycle C1 gases by converting them into value-added biochemicals using the Wood-Ljungdahl pathway. Despite the advantage of utilizing acetogens as biocatalysts, it is difficult to develop industrial-scale bioprocesses because of their slow growth rates and low productivities. To solve these problems, conventional approaches to metabolic engineering have been applied; however, there are several limitations owing to the lack of required genetic bioparts for regulating their metabolic pathways. Recently, synthetic biology based on genetic parts, modules, and circuit design has been actively exploited to overcome the limitations in acetogen engineering. This review covers synthetic biology applications to design and build industrial platform acetogens.

Gas seepage pockmark microbiomes suggest the presence of sedimentary coal seams in the Öxarfjörður graben of northeastern Iceland.

Natural gas seepage pockmarks are found off- and onshore in the Öxarfjörður graben, Iceland. The bacterial communities of two onshore seepage sites were analysed by 16S rRNA gene amplicon sequencing; the geochemical characteristics, hydrocarbon content, and the carbon isotope composition of the sites were also determined. While one site was found to be characterised by biogenic origin of methane gas, with a carbon isotope ratio (δ13C (‰)) of -63.2, high contents of organic matter and complex hydrocarbons, the other site showed a mixed origin of the methane gas (δ13C (‰) = -26.6) with geothermal characteristics and lower organic matter content. While both sites harboured Proteobacteria as the most abundant bacterial phyla, the Deltaproteobacteria were more abundant at the geothermal site and the Alphaproteobacteria at the biogenic site. The Dehalococcoidia class of phylum Chloroflexi was abundant at the geothermal site while the Anaerolineae class was more abundant at the biogenic site. Bacterial strains from the seepage pockmarks were isolated on a variety of selective media targeting bacteria with bioremediation potential. A total of 106 strains were isolated and characterised, including representatives from the phyla Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria. This article describes the first microbial study on gas seepage pockmarks in Iceland.

Novel facultative Methylocella strains are active methane consumers at terrestrial natural gas seeps.

BACKGROUND: Natural gas seeps contribute to global climate change by releasing substantial amounts of the potent greenhouse gas methane and other climate-active gases including ethane and propane to the atmosphere. However, methanotrophs, bacteria capable of utilising methane as the sole source of carbon and energy, play a significant role in reducing the emissions of methane from many environments. Methylocella-like facultative methanotrophs are a unique group of bacteria that grow on other components of natural gas (i.e. ethane and propane) in addition to methane but a little is known about the distribution and activity of Methylocella in the environment. The purposes of this study were to identify bacteria involved in cycling methane emitted from natural gas seeps and, most importantly, to investigate if Methylocella-like facultative methanotrophs were active utilisers of natural gas at seep sites. RESULTS: The community structure of active methane-consuming bacteria in samples from natural gas seeps from Andreiasu Everlasting Fire (Romania) and Pipe Creek (NY, USA) was investigated by DNA stable isotope probing (DNA-SIP) using 13C-labelled methane. The 16S rRNA gene sequences retrieved from DNA-SIP experiments revealed that of various active methanotrophs, Methylocella was the only active methanotrophic genus common to both natural gas seep environments. We also isolated novel facultative methanotrophs, Methylocella sp. PC1 and PC4 from Pipe Creek, able to utilise methane, ethane, propane and various non-gaseous multicarbon compounds. Functional and comparative genomics of these new isolates revealed genomic and physiological divergence from already known methanotrophs, in particular, the absence of mxa genes encoding calcium-containing methanol dehydrogenase. Methylocella sp. PC1 and PC4 had only the soluble methane monooxygenase (sMMO) and lanthanide-dependent methanol dehydrogenase (XoxF). These are the first Alphaproteobacteria methanotrophs discovered with this reduced functional redundancy for C-1 metabolism (i.e. sMMO only and XoxF only). CONCLUSIONS: Here, we provide evidence, using culture-dependent and culture-independent methods, that Methylocella are abundant and active at terrestrial natural gas seeps, suggesting that they play a significant role in the biogeochemical cycling of these gaseous alkanes. This might also be significant for the design of biotechnological strategies for controlling natural gas emissions, which are increasing globally due to unconventional exploitation of oil and gas.

Intramolecular isotopic evidence for bacterial oxidation of propane in subsurface natural gas reservoirs.

Microbial anaerobic oxidation of hydrocarbons is a key process potentially involved in a myriad of geological and biochemical environments yet has remained notoriously difficult to identify and quantify in natural environments. We performed position-specific carbon isotope analysis of propane from cracking and incubation experiments. Anaerobic bacterial oxidation of propane leads to a pronounced and previously unidentified 13C enrichment in the central position of propane, which contrasts with the isotope signature associated with the thermogenic process. This distinctive signature allows the detection and quantification of anaerobic oxidation of hydrocarbons in diverse natural gas reservoirs and suggests that this process may be more widespread than previously thought. Position-specific isotope analysis can elucidate the fate of natural gas hydrocarbons and provide insight into a major but previously cryptic process controlling the biogeochemical cycling of globally significant greenhouse gases.

High-throughput sequencing approach in analysis of microbial communities colonizing natural gas pipelines.

This study provides a deep modern insight into the phylogenetic diversity among bacterial consortia found in working and nonworking high-methane natural gas pipelines located in Poland. The working pipeline was characterized by lower biodiversity (140-154 bacterial genera from 22 to 23 classes, depending on the source of the debris) in comparison to the off-gas pipeline (169 bacterial genera from 23 classes). The sediment recovered from the working pipeline contained mostly DNA identified as belonging to the phylum Firmicutes (66.4%-45.9% operational taxonomic units [OTUs]), predominantly Bacillus (41.4%-31.1% OTUs) followed by Lysinibacillus (2.6%-1.5% OTUs) and Clostridium (2.4%-1.8% OTUs). In the nonworking pipeline, Proteobacteria (46.8% OTUs) and Cyanobacteria (27.8% OTUs) were dominant. Over 30% of the Proteobacteria sequences showed homologies to Gammaproteobacteria, with Pseudomonas (7.1%), Enhydrobacter (2.1%), Stenotrophomonas (0.5%), and Haempohilus (0.4%) among the others. Differences were noted in terms of the chemical compositions of deposits originating from the working and nonworking gas pipelines. The deposits from the nonworking gas pipeline contained iron, as well as carbon (42.58%), sulphur (15.27%), and oxygen (15.32%). This composition can be linked to both the quantity and type of the resident microorganisms. The presence of a considerable amount of silicon (17.42%), and of aluminum, potassium, calcium, and magnesium at detectable levels, may likewise affect the metabolic activity of the resident consortia in the working gas pipeline. All the analyzed sediments included both bacteria known for causing and intensifying corrosion (e.g., Pseudomonas, Desulfovibrio, Shewanella, Serratia) and bacteria that can protect the surface of pipelines against deterioration (e.g., Bacillus). Biocorrosion is not related to a single mechanism or one species of microorganism, but results from the multidirectional activity of multiple microbial communities. The analysis presented here of the state of the microbiome in a gas pipeline during the real gas transport is a particularly valuable element of this work.

Microbial community structure in deep natural gas-bearing aquifers subjected to sulfate-containing fluid injection.

In the natural gas field located in central Japan, high concentrations of natural gases and iodide ions are dissolved in formation water and commercially produced in deep aquifers. In the iodine recovery process, the produced formation water is amended with sulfate, and this fluid is injected into gas-bearing aquifers, which may lead to infrastructure corrosion by hydrogen sulfide. In this study, we examined the microbial community in aquifers subjected to sulfate-containing fluid injection. Formation water samples were collected from production wells located at different distances from the injection wells. The chemical analysis showed that the injection fluid contained oxygen, nitrate, nitrite and sulfate, in contrast to the formation water, which had previously been shown to be depleted in these components. Sulfur isotopic analysis indicated that sulfate derived from the injection fluid was present in the sample collected from near the injection wells. Quantitative and sequencing analysis of dissimilatory sulfite reductase and 16S rRNA genes revealed that sulfate-reducing bacteria (SRB), sulfur-oxidizing bacteria, and anaerobic methanotrophic archaea (ANME) in the wells located near injection wells were more abundant than those in wells located far from the injection wells, suggesting that fluid injection stimulated these microorganisms through the addition of oxygen, nitrate, nitrite and sulfate to the methane-rich aquifers. The predominant taxa were assigned to the ANME-2 group, its sulfate-reducing partner SEEP-SRB1 cluster and sulfur-oxidizing Epsilonproteobacteria. These results provide important insights for future studies to support the development of natural gas and iodine resources in Japan.

Recovering cellular biomass from fluids using chemical flocculation.

We developed an efficient, scalable and inexpensive method for recovering cellular biomass from complex fluid matrices that cannot be processed using conventional filtration methods. The method uses chemical flocculation with iron oxyhydroxides, is capable of recovering greater than 90% of cellular biomass from fluids with more than 103 cells ml-1 , and was validated using both mock communities and field samples. High quality DNA can be readily extracted from iron flocs using standard soil extraction kits. We applied chemical flocculation to fracing fluids from British Columbia, Canada and recovered a diversity of microbial taxa including abundant members of the Epsilon- and Deltaproteobacteria previously recovered from shale gas operations in the United States. Application of chemical flocculation presents new opportunities for scalable time-series monitoring and experimentation on complex fluid matrices including microbial community profiling and shotgun metagenomics over gas production well completion cycles.

Coupled laboratory and field investigations resolve microbial interactions that underpin persistence in hydraulically fractured shales.

Hydraulic fracturing is one of the industrial processes behind the surging natural gas output in the United States. This technology inadvertently creates an engineered microbial ecosystem thousands of meters below Earth's surface. Here, we used laboratory reactors to perform manipulations of persisting shale microbial communities that are currently not feasible in field scenarios. Metaproteomic and metabolite findings from the laboratory were then corroborated using regression-based modeling performed on metagenomic and metabolite data from more than 40 produced fluids from five hydraulically fractured shale wells. Collectively, our findings show that Halanaerobium, Geotoga, and Methanohalophilus strain abundances predict a significant fraction of nitrogen and carbon metabolites in the field. Our laboratory findings also exposed cryptic predatory, cooperative, and competitive interactions that impact microorganisms across fractured shales. Scaling these results from the laboratory to the field identified mechanisms underpinning biogeochemical reactions, yielding knowledge that can be harnessed to potentially increase energy yields and inform management practices in hydraulically fractured shales.

Facultative methanotrophs are abundant at terrestrial natural gas seeps.

BACKGROUND: Natural gas contains methane and the gaseous alkanes ethane, propane and butane, which collectively influence atmospheric chemistry and cause global warming. Methane-oxidising bacteria, methanotrophs, are crucial in mitigating emissions of methane as they oxidise most of the methane produced in soils and the subsurface before it reaches the atmosphere. Methanotrophs are usually obligate, i.e. grow only on methane and not on longer chain alkanes. Bacteria that grow on the other gaseous alkanes in natural gas such as propane have also been characterised, but they do not grow on methane. Recently, it was shown that the facultative methanotroph Methylocella silvestris grew on ethane and propane, other components of natural gas, in addition to methane. Therefore, we hypothesised that Methylocella may be prevalent at natural gas seeps and might play a major role in consuming all components of this potent greenhouse gas mixture before it is released to the atmosphere. RESULTS: Environments known to be exposed to biogenic methane emissions or thermogenic natural gas seeps were surveyed for methanotrophs. 16S rRNA gene amplicon sequencing revealed that Methylocella were the most abundant methanotrophs in natural gas seep environments. New Methylocella-specific molecular tools targeting mmoX (encoding the soluble methane monooxygenase) by PCR and Illumina amplicon sequencing were designed and used to investigate various sites. Functional gene-based assays confirmed that Methylocella were present in all of the natural gas seep sites tested here. This might be due to its ability to use methane and other short chain alkane components of natural gas. We also observed the abundance of Methylocella in other environments exposed to biogenic methane, suggesting that Methylocella has been overlooked in the past as previous ecological studies of methanotrophs often used pmoA (encoding the alpha subunit of particulate methane monooxygenase) as a marker gene. CONCLUSION: New biomolecular tools designed in this study have expanded our ability to detect, and our knowledge of the environmental distribution of Methylocella, a unique facultative methanotroph. This study has revealed that Methylocella are particularly abundant at natural gas seeps and may play a significant role in biogeochemical cycling of gaseous hydrocarbons.

Temporal dynamics of halogenated organic compounds in Marcellus Shale flowback.

The chemistry of hydraulic fracturing fluids and wastewaters is complex and is known to vary by operator, geologic formation, and fluid age. A time series of hydraulic fracturing fluids, flowback fluids, and produced waters was collected from two adjacent Marcellus Shale gas wells for organic chemical composition analyses using ultrahigh resolution mass spectrometry. Hierarchical clustering was used to compare and extract ions related to different fluid ages and many halogenated organic molecular ions were identified in flowback fluids and early produced waters based on exact mass. Iodinated organic compounds were the dominant halogen class in these clusters and were nearly undetectable in hydraulic fracturing fluid prior to injection. The iodinated ions increased in flowback and remained elevated after ten months of well production. We suggest that these trends are mainly driven by dissolved organic matter reacting with reactive halogen species formed abiotically through oxidizing chemical additives applied to the well and biotically via iodide-oxidizing bacteria. Understanding the implications of these identified halogenated organic compounds will require future investigation in to their structures and environmental fate.

Iodidimonas muriae gen. nov., sp. nov., an aerobic iodide-oxidizing bacterium isolated from brine of a natural gas and iodine recovery facility, and proposals of Iodidimonadaceae fam. nov., Iodidimonadales ord. nov., Emcibacteraceae fam. nov. and Emcibacterales ord. nov.

A chemo-organotrophic iodide (I-)-oxidizing bacterial strain, C-3T, isolated from natural gas brine of an iodine recovery facility in Kujukuri, Chiba, Japan, was characterized for representation of a novel species in the class Alphaproteobacteria. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the nearest neighbours of strain C-3T were members of the genera Eilatimonas, Kordiimonas, Rhodothalassium and Temperatibacter with 88-91 % sequence similarity. Cells of strain C-3T were aerobic, Gram-staining-negative, non-sporulating and rod-shaped (1.3-3.6 µm in length). Strain C-3T grew optimally at 30 °C, pH 7.5 and with 3 % NaCl (w/v). Iodide oxidation to form molecular iodine (I2) was a unique trait for strain C-3T, whereas the strain did not utilize iodide as a sole electron donor for chemolithoautotrophic growth. The major isoprenoid quinone was Q-10. The major cellular fatty acids were C18 : 1ω7c and C16 : 1ω5c. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol and unidentified aminolipids. The G+C content of the genomic DNA was 58.5 mol%. Iodide oxidation and the major cellular fatty acids composition distinguished strain C-3T from phylogenetically related bacteria. On the basis of the phenotypic features and the phylogenetic position, a novel genus and species are proposed for strain C-3T (=JCM 17843T=LMG 28660T), to be named Iodidimonas muriae gen. nov., sp. nov. We also propose to place the distinct sublineages of the genera Iodidimonasgen. nov. and Emcibacter in the orders Iodidimonadales ord. nov. and Emcibacterales ord. nov., respectively, because these genera are located far apart from the order Kordiimonadales and form the distinct lineage in the class Alphaproteobacteria.

The functional potential of microbial communities in hydraulic fracturing source water and produced water from natural gas extraction characterized by metagenomic sequencing.

Microbial activity in produced water from hydraulic fracturing operations can lead to undesired environmental impacts and increase gas production costs. However, the metabolic profile of these microbial communities is not well understood. Here, for the first time, we present results from a shotgun metagenome of microbial communities in both hydraulic fracturing source water and wastewater produced by hydraulic fracturing. Taxonomic analyses showed an increase in anaerobic/facultative anaerobic classes related to Clostridia, Gammaproteobacteria, Bacteroidia and Epsilonproteobacteria in produced water as compared to predominantly aerobic Alphaproteobacteria in the fracturing source water. The metabolic profile revealed a relative increase in genes responsible for carbohydrate metabolism, respiration, sporulation and dormancy, iron acquisition and metabolism, stress response and sulfur metabolism in the produced water samples. These results suggest that microbial communities in produced water have an increased genetic ability to handle stress, which has significant implications for produced water management, such as disinfection.

Methanohalophilus levihalophilus sp. nov., a slightly halophilic, methylotrophic methanogen isolated from natural gas-bearing deep aquifers, and emended description of the genus Methanohalophilus.

A mesophilic, slightly halophilic, obligately methylotrophic, methanogenic archaeon, designated strain GTA13(T), was isolated from natural gas-bearing confined aquifers in the Minami-Kanto gas field, Japan. The cells were non-motile, slightly irregular cocci, 0.7-1.0 µm in diameter and occurred singly, in pairs or as small aggregates. The cells grew with tri- or dimethylamine but not with H2/CO2, formate, acetate, methanol or dimethyl sulphide. Vitamins, sodium and magnesium were required for growth. Optimal growth occurred at pH 7.0-7.5, 35 °C, 0.35-0.40 M NaCl and 15-50 mM MgCl2. The NaCl range for growth was 0.2-1.3 M. The DNA G+C content was 43.7 mol%. Strain GTA13(T) showed highest levels of 16S rRNA gene sequence similarity with Methanohalophilus portucalensis FDF-1(T) (96.4% sequence similarity) and Methanohalophilus halophilus DSM 3094(T) (96.0%). On the basis of physiological and phylogenetic features, strain GTA13(T) is considered to represent a novel species of the genus Methanohalophilus, for which the name Methanohalophilus levihalophilus sp. nov. is proposed. The type strain is GTA13(T) ( = NBRC 110099(T) = DSM 28452(T)). An emended description of the genus Methanohalophilus is also proposed.

Viability and adaptation potential of indigenous microorganisms from natural gas field fluids in high pressure incubations with supercritical CO2.

Carbon Capture and Storage (CCS) is currently under debate as large-scale solution to globally reduce emissions of the greenhouse gas CO2. Depleted gas or oil reservoirs and saline aquifers are considered as suitable reservoirs providing sufficient storage capacity. We investigated the influence of high CO2 concentrations on the indigenous bacterial population in the saline formation fluids of a natural gas field. Bacterial community changes were closely examined at elevated CO2 concentrations under near in situ pressures and temperatures. Conditions in the high pressure reactor systems simulated reservoir fluids i) close to the CO2 injection point, i.e. saturated with CO2, and ii) at the outer boundaries of the CO2 dissolution gradient. During the incubations with CO2, total cell numbers remained relatively stable, but no microbial sulfate reduction activity was detected. After CO2 release and subsequent transfer of the fluids, an actively sulfate-respiring community was re-established. The predominance of spore-forming Clostridiales provided evidence for the resilience of this taxon against the bactericidal effects of supercritical (sc)CO2. To ensure the long-term safety and injectivity, the viability of fermentative and sulfate-reducing bacteria has to be considered in the selection, design, and operation of CCS sites.

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.

Methanothermobacter tenebrarum sp. nov., a hydrogenotrophic, thermophilic methanogen isolated from gas-associated formation water of a natural gas field.

A thermophilic and hydrogenotrophic methanogen, strain RMAS(T), was isolated from gas-associated formation water of a gas-producing well in a natural gas field in Japan. Strain RMAS(T) grew solely on H(2)/CO(2) but required Casamino acids, tryptone, yeast extract or vitamins for growth. Growth of strain RMAS(T) was stimulated by acetate. Cells were non-motile, straight rods (0.5×3.5-10.5 µm) and occurred singly or in pairs. Bundles of fimbriae occurred at both poles of cells and the cell wall was thick (approximately 21 nm, as revealed by ultrathin section electron microscopy). Strain RMAS(T) grew at 45-80 °C (optimum, 70 °C), at pH 5.8-8.7 (optimum, pH 6.9-7.7) and with 0.001-20 g NaCl l(-1) (optimum, 2.5 g NaCl l(-1)). Phylogenetic analysis revealed that Methanothermobacter thermautotrophicus ΔH(T) was most closely related to the isolate (95.7 % 16S rRNA gene sequence similarity). On the basis of morphological, phenotypic and phylogenetic characteristics, it is clear that strain RMAS(T) represents a novel species of the genus Methanothermobacter, for which we propose the name Methanothermobacter tenebrarum sp. nov. The type strain is RMAS(T) ( = DSM 23052(T) = JCM 16532(T) = NBRC 106236(T)).

Influence of the drilling mud formulation process on the bacterial communities in thermogenic natural gas wells of the Barnett Shale.

The Barnett Shale in north central Texas contains natural gas generated by high temperatures (120 to 150°C) during the Mississippian Period (300 to 350 million years ago). In spite of the thermogenic origin of this gas, biogenic sulfide production and microbiologically induced corrosion have been observed at several natural gas wells in this formation. It was hypothesized that microorganisms in drilling muds were responsible for these deleterious effects. Here we collected drilling water and drilling mud samples from seven wells in the Barnett Shale during the drilling process. Using quantitative real-time PCR and microbial enumerations, we show that the addition of mud components to drilling water increased total bacterial numbers, as well as the numbers of culturable aerobic heterotrophs, acid producers, and sulfate reducers. The addition of sterile drilling muds to microcosms that contained drilling water stimulated sulfide production. Pyrosequencing-based phylogenetic surveys of the microbial communities in drilling waters and drilling muds showed a marked transition from typical freshwater communities to less diverse communities dominated by Firmicutes and Gammaproteobacteria. The community shifts observed reflected changes in temperature, pH, oxygen availability, and concentrations of sulfate, sulfonate, and carbon additives associated with the mud formulation process. Finally, several of the phylotypes observed in drilling muds belonged to lineages that were thought to be indigenous to marine and terrestrial fossil fuel formations. Our results suggest a possible alternative exogenous origin of such phylotypes via enrichment and introduction to oil and natural gas reservoirs during the drilling process.