A contribution of hydrogenotrophic methanogenesis to the biogenic coal bed methane reserves of Southern Qinshui Basin,China

The activity of methanogens and related bacteria which inhabit the coal beds is essential for stimulating new biogenic coal bed methane (CBM) production from the coal matrix. In this study, the microbial community structure and methanogenesis were investigated in Southern Qinshui Basin in China, and the composition and stable isotopic ratios of CBM were also determined. Although geochemical analysis suggested a mainly thermogenic origin for CBM, the microbial community structure and activities strongly implied the presence of methanogens in situ. 454 pyrosequencing analysis combined with methyl coenzyme-M reductase (mcrA) gene clone library analysis revealed that the archaeal communities in the water samples from both coal seams were similar, with the dominance of hydrogenotrophic methanogen Methanobacterium. The activity and potential of these populations to produce methane were confirmed by the observation of methane production in enrichments supplemented with H₂ + CO₂ and formate, and the only archaea successfully propagated in the tested water samples was from the genus Methanobacterium. 454 pyrosequencing analysis also recovered the diverse bacterial communities in the water samples, which have the potential to play a role in the coal biodegradation fueling methanogens. These results suggest that the biogenic CBM was generated by coal degradation via the hydrogenotrophic methanogens and related bacteria, which also contribute to the huge CBM reserves in Southern Qinshui Basin, China.     

芦岭煤田微生物群落结构和生物成因气的产甲烷类型研究

【目的】揭示芦岭煤田微生物群落组成,并分析其潜在的产甲烷类型及产甲烷途径。【方法】采集芦岭煤田的煤层气样品和产出水样品,分别分析样品的地球化学性质特征;利用Illumina HiSeq高通量测序技术分析产出水中的微生物群落结构;采用添加不同底物的厌氧培养实验进一步证实芦岭煤田生物成因气的产甲烷类型。【结果】该地区煤层气为生物成因和热成因的混合成因气;古菌16S rRNA基因分析表明在产出水中含有乙酸营养型、氢营养型和甲基营养型的产甲烷菌。丰度较高的细菌具有降解煤中芳香族和纤维素衍生化合物的潜力。厌氧富集培养结果表明,添加乙酸盐、甲酸盐、H2+CO2为底物的矿井水样均有明显的甲烷产生。【结论】芦岭煤田具有丰富的生物多样性,该地区同时存在三种产甲烷类型。本研究为利用微生物技术提高煤层气的采收率,实现煤层气的可持续开采提供科学依据。     

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.     

寺河矿煤地质产甲烷微生物菌群的保藏和产甲烷性能

【背景】煤地质产甲烷微生物菌群可以代谢煤基质产生甲烷,对于实现煤层气资源的再利用具有重要意义。【目的】检测产甲烷菌群在保藏过程中群落结构的动态变化以及在产气实验中甲烷气的生成情况,以验证保藏方法的可行性,同时为煤层气的微生物增产奠定基础。【方法】分别于不同温度条件下比较3种菌种保藏方法,即甘油/L-半胱氨酸法、富营养法和煤基-基础盐法。通过产气实验检测不同保藏条件下产甲烷菌群的活力。同时,采用454高通量测序技术测定16S r RNA基因序列,分析25°C条件下煤基-基础盐菌种保藏过程中微生物群落结构的变化。【结果】比较了9组菌种保藏方法,发现菌种最佳保藏条件为25°C的煤基-基础盐保藏。在该条件下保藏的产甲烷菌群活性最高,甲烷生成量最大。以无烟煤为碳源进行产气实验时甲烷生成量为12%-25%,而以褐煤为碳源时甲烷生成量可达24%-73%。在25°C的煤基-基础盐菌种保藏条件下,保藏初期细菌的主要优势菌为假单胞菌属(Pseudomonas),而古菌的主要优势菌为甲烷八叠球菌属(Methanosarcina)。随着保藏时间的增加,细菌的群落结构变化显著,发酵细菌及产氢产乙酸细菌成为优势细菌,古菌的群落结构则相对稳定。【结论】菌种保藏的最佳条件为25°C的煤基-基础盐,保藏的产甲烷菌群能长期维持在较高的活性状态,具有较好的产甲烷能力。     

Molecular characterization of microbial communities in deep coal seam groundwater of northern Japan

We investigated microbial methanogenesis and community structure based on 16S rRNA gene sequences from a coal seam aquifer located 843–907 m below ground level in northern Japan; additionally, we studied the δ¹³C and δ²H (δD) of coal‐bed gases and other physicochemical parameters. Although isotopic analysis suggested a thermocatalytic origin for the gases, the microbial activity and community structure strongly implied the existence of methanogenic microbial communities in situ. Methane was generated in the enrichment cultures of the hydrogenotrophic and methylotrophic microorganisms obtained from coal seam groundwater. Methanogen clones dominated the archaeal 16S rRNA gene libraries and were mostly related to the hydrogenotrophic genus Methanoculleus and the methylotrophic genus Methanolobus. Bacterial 16S rRNA gene libraries were dominated by the clones related to the genera Acetobacterium and Syntrophus which have a symbiotic association with methanogens. LIBSHUFF analysis revealed that N₂ gas injected into the coal seam (for enhanced methane production) does not affect the coverage of archaeal and bacterial populations. However, amova analysis does provide evidence for a change in the genetic diversity of archaeal populations that are dominated by methanogens. Therefore, N₂ injection into the coal seam might affect the cycling of matter by methanogens in situ.     

Evidence of Active Methanogen Communities in Shallow Sediments of the Sonora Margin Cold Seeps

In the Sonora Margin cold seep ecosystems (Gulf of California), sediments underlying microbial mats harbor high biogenic methane concentrations, fueling various microbial communities, such as abundant lineages of anaerobic methanotrophs (ANME). However, the biodiversity, distribution, and metabolism of the microorganisms producing this methane remain poorly understood. In this study, measurements of methanogenesis using radiolabeled dimethylamine, bicarbonate, and acetate showed that biogenic methane production in these sediments was mainly dominated by methylotrophic methanogenesis, while the proportion of autotrophic methanogenesis increased with depth. Congruently, methane production and methanogenic Archaea were detected in culture enrichments amended with trimethylamine and bicarbonate. Analyses of denaturing gradient gel electrophoresis (DGGE) fingerprinting and reverse-transcribed PCR-amplified 16S rRNA sequences retrieved from these enrichments revealed the presence of active methylotrophic Methanococcoides burtonii relatives and several new autotrophic Methanogenium lineages, confirming the cooccurrence of Methanosarcinales and Methanomicrobiales methanogens with abundant ANME populations in the sediments of the Sonora Margin cold seeps.     

Cultivation-independent analysis of archaeal and bacterial communities of the formation water in an Indian coal bed to enhance biotransformation of coal into methane

Biogenic origin of the significant proportion of coal bed methane has indicated the role of microbial communities in methanogenesis. By using cultivation-independent approach, we have analysed the archaeal and bacterial community present in the formation water of an Indian coal bed at 600–700 m depth to understand their role in methanogenesis. Presence of methanogens in the formation water was inferred by epifluorescence microscopy and PCR amplification of mcrA gene. Archaeal 16S rRNA gene clone library from the formation water metagenome was dominated by methanogens showing similarity to Methanobacterium, Methanothermobacter and Methanolinea whereas the clones of bacterial 16S rRNA gene library were closely related to Azonexus, Azospira, Dechloromonas and Thauera. Thus, microbial community of the formation water consisted of predominantly hydrogenotrophic methanogens and the proteobacteria capable of nitrogen fixation, nitrate reduction and polyaromatic compound degradation. Methanogenic potential of the microbial community present in the formation water was elucidated by the production of methane in the enrichment culture, which contained 16S rRNA gene sequences showing close relatedness to the genus Methanobacterium. Microcosm using formation water as medium as well as a source of inoculum and coal as carbon source produced significant amount of methane which increased considerably by the addition of nitrite. The dominance of Diaphorobacter sp. in nitrite amended microcosm indicated their important role in supporting methanogenesis in the coal bed. This is the first study indicating existence of methanogenic and bacterial community in an Indian coal bed that is capable of in situ biotransformation of coal into methane.     

Methane-producing microbial community in a coal bed of the Illinois basin

A series of molecular and geochemical studies were performed to study microbial, coal bed methane formation in the eastern Illinois Basin. Results suggest that organic matter is biodegraded to simple molecules, such as H(2) and CO(2), which fuel methanogenesis and the generation of large coal bed methane reserves. Small-subunit rRNA analysis of both the in situ microbial community and highly purified, methanogenic enrichments indicated that Methanocorpusculum is the dominant genus. Additionally, we characterized this methanogenic microorganism using scanning electron microscopy and distribution of intact polar cell membrane lipids. Phylogenetic studies of coal water samples helped us develop a model of methanogenic biodegradation of macromolecular coal and coal-derived oil by a complex microbial community. Based on enrichments, phylogenetic analyses, and calculated free energies at in situ subsurface conditions for relevant metabolisms (H(2)-utilizing methanogenesis, acetoclastic methanogenesis, and homoacetogenesis), H(2)-utilizing methanogenesis appears to be the dominant terminal process of biodegradation of coal organic matter at this location.     

基于mcrA基因的沁水盆地煤层气田产甲烷菌群与途径分析

【目的】分析沁水盆地煤层气田不同煤层气井产出水样中产甲烷菌群和生物成因气的生成途径。【方法】以甲基辅酶M还原酶基因(mcr A)作为目标基因,采用454焦磷酸高通量测序方法,同时比对NCBI功能基因文库中的mcr A序列,分析不同煤层气井产出水中的产甲烷菌群。【结果】高通量测序表明,5个出水样产甲烷菌群OTUs(Operational taxonomic units)数为64–157个,共有的为22个,各占样品总数14%-34%;样品共检测到4种已知菌属,即甲烷杆菌属(Methanobacterium)、甲烷微菌属(Methanomicrobium)、甲烷叶菌属(Methanolobus)和甲烷螺菌属(Methanospirillum),优势菌属均为Methanobacterium。系统发育分析表明,未明确地位的菌属主要与Methanobacterium、Methanomicrobium、产甲烷球菌属(Methanococcus)和甲烷囊菌属(Methanoculleus)有较近的亲缘关系。5个样品中菌属所占比例不同,检测到的菌属类别大致相同。所有检测样品生物成因煤层气(Coalbed methane,CBM)的生成途径主要为氢营养型产甲烷途径。【结论】沁水盆地不同煤层气田产甲烷菌群菌种差异比较大,但生物成因气生成途径基本相似,与地理位置和煤藏条件没有相关性。     

The influence of hydrogeological disturbance and mining on coal seam microbial communities

The microbial communities present in two underground coal mines in the Bowen Basin, Queensland, Australia, were investigated to deduce the effect of pumping and mining on subsurface methanogens and methanotrophs. The micro‐organisms in pumped water from the actively mined areas, as well as, pre‐ and post‐mining formation waters were analyzed using 16S rRNA gene amplicon sequencing. The methane stable isotope composition of Bowen Basin coal seam indicates that methanogenesis has occurred in the geological past. More recently at the mine site, changing groundwater flow dynamics and the introduction of oxygen in the subsurface has increased microbial biomass and diversity. Consistent with microbial communities found in other coal seam environments, pumped coal mine waters from the subsurface were dominated by bacteria belonging to the genera Pseudomonas and the family Rhodocyclaceae. These environments and bacterial communities supported a methanogen population, including Methanobacteriaceae, Methanococcaceae and Methanosaeta. However, one of the most ubiquitous micro‐organisms in anoxic coal mine waters belonged to the family ‘Candidatus Methanoperedenaceae’. As the Archaeal family ‘Candidatus Methanoperedenaceae’ has not been extensively defined, the one studied species in the family is capable of anaerobic methane oxidation coupled to nitrate reduction. This introduces the possibility that a methane cycle between archaeal methanogenesis and methanotrophy may exist in the anoxic waters of the coal seam after hydrogeological disturbance.     

Diversity in methane enrichments from agricultural soil revealed by DGGE separation of PCR amplified 16S rDNA fragments

Denaturing gradient gel electrophoresis (DGGE) profiles of PCR amplified V3 regions of 16S rRNA genes were used to assess the diversity in enrichment cultures with methane as the only carbon and energy source. The enrichments originated from two agricultural soils. One was a sandy soil with low (10%) organic content, the other an organic soil with approximately 50% organic content. DGGE provided a fast evaluation of the distribution of amplifiable sequence types indicating that specific bacterial populations had been enriched from each soil. The DGGE profiles revealed a broader range of amplified V3 fragments in the community derived from organic soil than from sandy soil. Fragments from 19 individual DGGE bands were sequenced and compared with 27 previously published 16S rRNA gene sequences. The sequences confirmed the high diversity with the presence of different methylotrophic populations in each enrichment. No affiliation was found with type I methanotrophs, instead type II methanotroph sequences were found in the enrichments from both soil types. Some of the fragments from the organic soil enrichment were not affiliated with methylotrophs. Most of the sequences clustered distantly on a branch within the α-Proteobacteria. These facts suggested that previously undescribed methylotrophs are abundant in methane enrichments from agricultural soil.     

胜利油田单12高温油藏非培养和富集培养样品的细菌组成特性

[目的]通过比较分析油藏样品的微生物群落结构特点,认识油藏微生物的生态功能.[方法]利用3种油藏微生物研究中常用的富集培养方法,对胜利油田单12区块S12-4油井产出水样品进行了选择性富集培养,运用构建16S rRNA基因文库的方法分析了富集样品和非培养样品的细菌多样性.[结果]通过16S rRNA基因序列比对发现,非培养样品、异养菌富集样品、烃降解菌富集样品和硫酸盐还原菌富集样品中的优势菌分别为Pseudomonas属,Thermotoga属,Thermaerobacter属和Thermotoga属的成员.多样性分析结果表明,非培养样品的微生物多样性最丰富,同时非培养样品和富集样品的微生物群落结构存在很大的差异,富集样品中的微生物包括优势菌在油藏原位环境中含量很低.[结论]细菌组成差异的比较结果,对油藏微生物的生态功能研究和微生物驱油潜力评估具有重要意义.     

Biogenic methane production in formation waters from a large gas field in the North Sea

Methanogenesis was investigated in formation waters from a North Sea oil rimmed gas accumulation containing biodegraded oil, which has not been subject to seawater injection. Activity and growth of hydrogenotrophic methanogens was measured but acetoclastic methanogenesis was not detected. Hydrogenotrophic methanogens showed activity between 40 and 80°C with a temperature optimum (ca. 70°C) consistent with in situ reservoir temperatures. They were also active over a broad salinity range, up to and consistent with the high salinity of the waters (90 g l⁻¹). These findings suggest the methanogens are indigenous to the reservoir. The conversion of H₂ and CO₂ to CH₄ in methanogenic enrichments was enhanced by the addition of inorganic nutrients and was correlated with cell growth. Addition of yeast extract also stimulated methanogenesis. Archaeal 16S rRNA gene sequences recovered from enrichment cultures were closely related to Methanothermobacter spp. which have been identified in other high-temperature petroleum reservoirs. It has recently been suggested that methanogenic oil degradation may be a major factor in the development of the world’s heavy oils and represent a significant and ongoing process in conventional deposits. Although an oil-degrading methanogenic consortium was not enriched from these samples the presence and activity of communities of fermentative bacteria and methanogenic archaea was demonstrated. Stimulation of methanogenesis by addition of nutrients suggests that in situ methanogenic biodegradation of oil could be harnessed to enhance recovery of stranded energy assets from such petroleum systems.     

Potential of biogenic methane for pilot-scale fermentation ex situ with lump anthracite and the changes of methanogenic consortia

Pilot-scale fermentation is one of the important processes for achieving industrialization of biogenic coalbed methane (CBM), although the mechanism of biogenic CBM remains unknown. In this study, 16 samples of formation water from CBM production wells were collected and enriched for methane production, and the methane content was between 3.1 and 21.4%. The formation water of maximum methane production was used as inoculum source for pilot-scale fermentation. The maximum methane yield of the pilot-scale fermentation with lump anthracite amendment reached 13.66 μmol CH₄/mL, suggesting that indigenous microorganisms from formation water degraded coal to produce methane. Illumina high-throughput sequencing analysis revealed that the bacterial and archaeal communities in the formation water sample differed greatly from the methanogic water enrichment culture. The hydrogenotrophic methanogen Methanocalculus dominated the formation water. Acetoclastic methanogens, from the order Methanosarcinales, dominated coal bioconversion. Thus, the biogenic methanogenic pathway ex situ cannot be simply identified according to methanogenic archaea in the original inoculum. Importantly, this study was the first time to successfully simulate methanogenesis in large-capacity fermentors (160 L) with lump anthracite amendment, and the result was also a realistic case for methane generation in pilot-scale ex situ.     

Methanogenesis from methanol at low temperatures by a novel psychrophilic methanogen, "Methanolobus psychrophilus" sp. nov., prevalent in Zoige wetland of the Tibetan plateau

The Zoige wetland of the Tibetan plateau is at permanent low temperatures and is a methane emission heartland of the plateau; however, cold-adaptive methanogens in the soil are poorly understood. In this study, a variety of methanogenic enrichments at 15 degrees C and 30 degrees C were obtained from the wetland soil. It was demonstrated that hydrogenotrophic methanogenesis was the most efficient type at 30 degrees C, while methanol supported the highest methanogenesis rate at 15 degrees C. Moreover, methanol was the only substrate to produce methane more efficiently at 15 degrees C than at 30 degrees C. A novel psychrophilic methanogen, strain R15, was isolated from the methanol enrichment at 15 degrees C. Phylogenetic analysis placed strain R15 within the genus Methanolobus, loosely clustered with Methanolobus taylorii (96.7% 16S rRNA similarity). R15 produced methane from methanol, trimethylamine, and methyl sulfide and differed from other Methanolobus species by growing and producing methane optimally at 18 degrees C (specific growth rate of 0.063 +/- 0.001 h(-1)) and even at 0 degrees C. Based on these characteristics, R15 was proposed to be a new species and named "Methanolobus psychrophilus" sp. nov. The K(m) and V(max) of R15 for methanol conversion were determined to be 87.5 +/- 0.4 microM and 0.39 +/- 0.04 mM h(-1) at 18 degrees C, respectively, indicating a high affinity and conversion efficiency for methanol. The proportion of R15 in the soil was determined by quantitative PCR, and it accounted for 17.2% +/- 2.1% of the total archaea, enumerated as 10(7) per gram of soil; the proportion was increased to 42.4% +/- 2.3% in the methanol enrichment at 15 degrees C. This study suggests that the psychrophilic methanogens in the Zoige wetland are likely to be methylotrophic and to play a role in methane emission of the wetland.     

16S ribosomal RNA sequence analysis for determination of phylogenetic relationship among methylotrophs.

16S ribosomal RNAs (rRNA) of 12 methylotrophic bacteria have been almost completely sequenced to establish their phylogenetic relationships. Methylotrophs that are physiologically related are phylogenetically diverse and are scattered among the purple eubacteria (class Proteobacteria). Group I methylotrophs can be classified in the beta- and the gamma-subdivisions and group II methylotrophs in the alpha-subdivision of the purple eubacteria, respectively. Pink-pigmented facultative and non-pigmented obligate group II methylotrophs form two distinctly separate branches within the alpha-subdivision. The secondary structures of the 16S rRNA sequences of 'Methylocystis parvus' strain OBBP, 'Methylosinus trichosporium' strain OB3b, 'Methylosporovibrio methanica' strain 81Z and Hyphomicrobium sp. strain DM2 are similar, and these non-pigmented obligate group II methylotrophs form one tight cluster in the alpha-subdivision. The pink-pigmented facultative methylotrophs, Methylobacterium extorquens strain AM1, Methylobacterium sp. strain DM4 and Methylobacterium organophilum strain XX form another cluster within the alpha-subdivision. Although similar in phenotypic characteristics, Methylobacterium organophilum strain XX and Methylobacterium extorquens strain AM1 are clearly distinguishable by their 16S rRNA sequences. The group I methylotrophs, Methylophilus methylotrophus strain AS1 and methylotrophic species DM11, which do not utilize methane, are similar in 16S rRNA sequence to bacteria in the beta-subdivision. The methane-utilizing, obligate group I methanotrophs, Methylococcus capsulatus strain BATH and Methylomonas methanica, are placed in the gamma-subdivision. The results demonstrate that it is possible to distinguish and classify the methylotrophic bacteria using 16S rRNA sequence analysis.     

Persistence of Halophylic Methanogens and Oil-Degrading Bacteria in an Offshore Oil-Producing Facility

Microbial communities in a high saline, Tetrakis-Hydroxymethyl Phosphonium Sulfate (THPS) and nitrate-treated Nigerian oil-producing facilities were investigated. Methanogens in produced water samples preferred methanol, while those in pig-run samples (oily wastes from pipelines) preferred H₂/CO₂, as substrates to produce methane and stimulate metal corrosion. The results coincide with the dominance of methylotrophic and hydrogenotrophic methanogens in the respective samples. The same microbial populations were also THPS and high salinity tolerant. The nitrate reducers and hydrocarbon degraders were also dominant in the reservoir. A more inclusive and effective mitigation strategy is therefore required to effectively tackle biocide resistant methanogens in biocide treated oilfield.     

The impact of aridification and vegetation type on changes in the community structure of methane-cycling microorganisms in Japanese wetland soils

Over the years, the wetlands covered by Sphagnum in Bibai, Japan have been turning into areas of aridity, resulting in an invasion of Sasa into the bogs. Yet little is known about the methane-cycling microorganisms in such environments. In this study, the methanotrophic, methanogenic, and archaeal community structures within these two types of wetland vegetation were studied by phylogenetic analysis targeting particulate methane monooxygenase (pmoA), methyl coenzyme M reductase (mcrA), and the archaeal 16S rRNA gene. The pmoA library indicated that Methylomonas and Methylocystis predominated in the Sphagnum-covered and Sasa-invaded areas, respectively. The mcrA and 16S rRNA libraries indicated that Methanoregula were abundant methanogens in the Sphagnum-covered area. In the Sasa-invaded area, by contrast, mcrA genes were not detected, and no 16S rRNA clones were affiliated with previously known methanogens. Because the Sasa-invaded area still produced methane, of the various uncultured populations detected, novel euryarchaeotal lineages are candidate methane producers.     

Salinity constraints on subsurface archaeal diversity and methanogenesis in sedimentary rock rich in organic matter

The diversity of microorganisms active within sedimentary rocks provides important controls on the geochemistry of many subsurface environments. In particular, biodegradation of organic matter in sedimentary rocks contributes to the biogeochemical cycling of carbon and other elements and strongly impacts the recovery and quality of fossil fuel resources. In this study, archaeal diversity was investigated along a salinity gradient spanning 8 to 3,490 mM Cl(-) in a subsurface shale rich in CH(4) derived from biodegradation of sedimentary hydrocarbons. Shale pore waters collected from wells in the main CH(4)-producing zone lacked electron acceptors such as O(2), NO(3)(-), Fe(3+), or SO(4)(2-). Acetate was detected only in high-salinity waters, suggesting that acetoclastic methanogenesis is inhibited at Cl(-) concentrations above approximately 1,000 mM. Most-probable-number series revealed differences in methanogen substrate utilization (acetate, trimethylamine, or H(2)/CO(2)) associated with chlorinity. The greatest methane production in enrichment cultures was observed for incubations with salinity at or close to the native pore water salinity of the inoculum. Restriction fragment length polymorphism analyses of archaeal 16S rRNA genes from seven wells indicated that there were links between archaeal communities and pore water salinity. Archaeal clone libraries constructed from sequences from 16S rRNA genes isolated from two wells revealed phylotypes similar to a halophilic methylotrophic Methanohalophilus species and a hydrogenotrophic Methanoplanus species at high salinity and a single phylotype closely related to Methanocorpusculum bavaricum at low salinity. These results show that several distinct communities of methanogens persist in this subsurface, CH(4)-producing environment and that each community is adapted to particular conditions of salinity and preferential substrate use and each community induces distinct geochemical signatures in shale formation waters.     

Substrate Availability,pH, and Temperature Influence Methanogenesis and Mild Steel Corrosion

Solid deposits of corroded pipelines in the Niger Delta were analyzed both chemically and microbiologically. The addition of substrate, especially acetate, significantly stimulated the methane production ranging from 0.85 to 1.60 mmole compared with 0.65 mmole of the control. Acetotrophic and hydrogenotrophic methanogens had their optimal methane production and corrosion rate at pH 5.5. All three types of methanogens produced the most methane at 37°C. Methane production by methanogens correlated strongly with corrosion rate. Pyrosequencing surveys show dominance of acetotrophic, hydrogenotrophic, and methylotrophic methanogens in the samples with no significant presence of sulfate-reducing bacteria (SRB), most likely due to the presence of the biocide, tetrakis-hydroxymethyl phosphonium sulfate. Our study shows that methanogens were one possible cause of pipeline failures in samples from the Niger Delta without the syntrophic association with SRB.