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.     

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。     

Methylotrophic methanogenesis governs the biogenic coal bed methane formation in Eastern Ordos Basin, China

To identify the methanogenic pathways present in a deep coal bed methane (CBM) reservoir associated with Eastern Ordos Basin in China, a series of geochemical and microbiological studies was performed using gas and water samples produced from the Liulin CBM reservoir. The composition and stable isotopic ratios of CBM implied a mixed biogenic and thermogenic origin of the methane. Archaeal 16S rRNA gene analysis revealed the dominance of the methylotrophic methanogen Methanolobus in the water produced. The high potential of methane production by methylotrophic methanogens was found in the enrichments using the water samples amended with methanol and incubated at 25 and 35?°C. Methylotrophic methanogens were the dominant archaea in both enrichments as shown by polymerase chain reaction (PCR)–denaturing gradient gel electrophoresis (DGGE). Bacterial 16S rRNA gene analysis revealed that fermentative, sulfate-reducing, and nitrate-reducing bacteria inhabiting the water produced were a factor in coal biodegradation to fuel methanogens. These results suggested that past and ongoing biodegradation of coal by methylotrophic methanogens and syntrophic bacteria, as well as thermogenic CBM production, contributed to the Liulin CBM reserves associated with the Eastern Ordos Basin.     

不同煤阶生物成因煤层气微生物群落的功能及多样性研究进展

生物成因煤层气是煤层气形成的主要途径之一,在各种煤阶的煤层气田中均发现有生物成因煤层气。高通量测序技术、宏基因组学等高新技术的应用逐步揭示了生物成因煤层气微生物群落的组成及多样性,为揭示煤层气资源的微生物生成机理提供了理论依据。本文综述了近年来国内外针对不同煤阶条件下微生物群落结构及多样性方面的研究进展,总结了煤层气生物成因过程中主要微生物的功能及产气途径,并探讨了生物成因煤层气领域的研究前景。     

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.     

微生物增产煤层气技术研究进展

微生物增产煤层气技术能够产生新的煤层气,有效缓解能源紧张问题,是煤层气开采、增产的研究热点。微生物增产煤层气技术的核心机理是煤的厌氧生物降解产甲烷。国内外研究学者通过实验室模拟研究了本源和外源微生物的降解煤产甲烷能力及影响因素。部分煤层气公司富集或分离获得了高效产甲烷菌群,并研究了微生物增产煤层气技术的现场实施方法。开发煤的预处理技术、构建新型高效菌群等可进一步提高微生物的增产煤层气能力,促进微生物增产煤层气技术的应用。     

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。     

The characteristics and comparative analysis of methanotrophs reveal genomic insights into Methylomicrobium sp. enriched from marine sediments

Methanotrophic bacteria are widespread and use methane as a sole carbon and energy source. They also play a crucial role in marine ecosystems by preventing the escape of methane into the atmosphere from diverse methane sources, such as methane seeps and hydrothermal vents. Despite their importance for methane carbon cycling, relatively few marine methanotrophic bacteria have been isolated and studied at the genomic level. Herein, we report the genome of a marine methanotrophic member of the genus Methylomicrobium, metagenome-assembled genome (MAG) wino1, which was obtained through enrichment using methane as the sole carbon source. Phylogenetic analysis based on 16S rRNA sequences and comparison of pmoA genes supported the close relationship of MAG-wino1 to the genus Methylomicrobium and it possessed a genome of 5.06 Mb encoding many specialized methanotrophic genes. A comparison of MAG-wino1 with the genomes of other strains (Methylomicrobium alcaliphilum 20Z^T and Methylomicrobium buryatense 5G) showed that genes (e.g. ectABC, ask, and mscLS) involved in the accumulation of compatible solutes required for survival in marine environments might be conserved. Methane utilization genes, including methanol dehydrogenase, and key enzymes related to ribulose monophosphate (RuMP) metabolism were identified. The wino1 genome harbored nitrogen fixation, urease, urea and nitrate transporter genes involved in the exploitation of nitrogen sources. Poly-β-hydroxybutyrate degradation and glycogen synthesis-related genes may facilitate survival under nutrient-limiting conditions. Additionally, genome analysis revealed three dominant taxa in the enrichment culture, methanotroph Methylomicrobium sp., methylotroph Methyloceanibacter sp., and non-methylotroph Labrenzia sp., which provided insights into microbial associations in marine sediments.     

Phylogenetic and Functional Diversity of Microbial Communities Associated with Subsurface Sediments of the Sonora Margin,Guaymas Basin

Subsurface sediments of the Sonora Margin (Guaymas Basin), located in proximity of active cold seep sites were explored. The taxonomic and functional diversity of bacterial and archaeal communities were investigated from 1 to 10 meters below the seafloor. Microbial community structure and abundance and distribution of dominant populations were assessed using complementary molecular approaches (Ribosomal Intergenic Spacer Analysis, 16S rRNA libraries and quantitative PCR with an extensive primers set) and correlated to comprehensive geochemical data. Moreover the metabolic potentials and functional traits of the microbial community were also identified using the GeoChip functional gene microarray and metabolic rates. The active microbial community structure in the Sonora Margin sediments was related to deep subsurface ecosystems (Marine Benthic Groups B and D, Miscellaneous Crenarchaeotal Group, Chloroflexi and Candidate divisions) and remained relatively similar throughout the sediment section, despite defined biogeochemical gradients. However, relative abundances of bacterial and archaeal dominant lineages were significantly correlated with organic carbon quantity and origin. Consistently, metabolic pathways for the degradation and assimilation of this organic carbon as well as genetic potentials for the transformation of detrital organic matters, hydrocarbons and recalcitrant substrates were detected, suggesting that chemoorganotrophic microorganisms may dominate the microbial community of the Sonora Margin subsurface sediments.     

Methane-fed microbial microcosms show differential community dynamics and pinpoint taxa involved in communal response

We report observations on the dynamics of bacterial communities in response to methane stimulus in laboratory microcosm incubations prepared with lake sediment samples. We first measured taxonomic compositions of long-term enrichment cultures and determined that, although dominated by Methylococcaceae types, these cultures also contained accompanying types belonging to a limited number of bacterial taxa, methylotrophs and non-methylotrophs. We then followed the short-term community dynamics, in two oxygen tension regimens (150 μM and 15 μM), observing rapid loss of species diversity. In all microcosms, a single type of Methylobacter represented the major methane-oxidizing partner. The accompanying members of the communities revealed different trajectories in response to different oxygen tensions, with Methylotenera species being the early responders to methane stimulus under both conditions. The communities in both conditions were convergent in terms of their assemblage, suggesting selection for specific taxa. Our results support prior observations from metagenomics on distribution of carbon from methane among diverse bacterial populations and further suggest that communities are likely responsible for methane cycling, rather than a single type of microbe.     

Phenotypic and genomic characterization of the Antarctic bacterium Gillisia sp. CAL575, a producer of antimicrobial compounds

Microorganisms from Antarctica have evolved particular strategies to cope with cold. Moreover, they have been recently reported as producers of antimicrobial compounds, which inhibit the growth of other bacteria. In this work we characterized from different viewpoints the Gillisia sp. CAL575 strain, a psychrotrophic bacterium that produces microbial volatile organic compounds involved in the growth inhibition of Burkholderia cepacia complex members. Sequencing and analysis of the whole genome of Gillisia sp. CAL575 revealed that it includes genes that are involved in secondary metabolite production, adaptation to cold conditions, and different metabolic pathways for the production of energy. All these features make Gillisia sp. CAL575 a possible tool for biotechnology.     

Enrichment and characteristics of mixed methane-oxidizing bacteria from a Chinese coal mine

In methane-rich environments, methane-oxidizing bacteria usually occur predominantly among consortia including other types of microorganisms. In this study, artificial coal bed gas and methane gas were used to enrich mixed methanotrophic cultures from the soil of a coal mine in China, respectively. The changes in microbial community structure and function during the enrichment were examined. The microbial diversity was reduced as the enrichment proceeded, while the capacity for methane oxidation was significantly enhanced by the increased abundance of methanotrophs. The proportion of type II methanotrophs increased greatly from 7.84 % in the sampled soil to about 50 % in the enrichment cultures, due to the increase of methane concentration. After the microbial community of the cultures got stable, Methylomonas and Methylocystis became the dominant type I and type II methanotrophs, while Methylophilus was the prevailing methylotroph. The sequences affiliated with pigment-producing strains, Methylomonas rubra, Hydrogenophaga sp. AH-24, and Flavobacterium cucumis, could explain the orange appearance of the cultures. Comparing the two cultures, the multi-carbon sources in the artificial coal bed gas caused more variety of non-methanotrophic bacteria, but did not help to maintain the diversity or to increase the quantity and activity of methanotrophs. The results could help to understand the succession and interaction of microbial community in a methane-driven ecosystem.     

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.     

Long-Term Enrichment on Cellulose or Xylan Causes Functional and Taxonomic Convergence of Microbial Communities from Anaerobic Digesters

Cellulose and xylan are two major components of lignocellulosic biomass, which represents a potentially important energy source, as it is abundant and can be converted to methane by microbial action. However, it is recalcitrant to hydrolysis, and the establishment of a complete anaerobic digestion system requires a specific repertoire of microbial functions. In this study, we maintained 2-year enrichment cultures of anaerobic digestion sludge amended with cellulose or xylan to investigate whether a cellulose- or xylan-digesting microbial system could be assembled from sludge previously used to treat neither of them. While efficient methane-producing communities developed under mesophilic (35°C) incubation, they did not under thermophilic (55°C) conditions. Illumina amplicon sequencing results of the archaeal and bacterial 16S rRNA genes revealed that the mature cultures were much lower in richness than the inocula and were dominated by single archaeal (genus Methanobacterium) and bacterial (order Clostridiales) groups, although at finer taxonomic levels the bacteria were differentiated by substrates. Methanogenesis was primarily via the hydrogenotrophic pathway under all conditions, although the identity and growth requirements of syntrophic acetate-oxidizing bacteria were unclear. Incubation conditions (substrate and temperature) had a much greater effect than inoculum source in shaping the mature microbial community, although analysis based on unweighted UniFrac distance found that the inoculum still determined the pool from which microbes could be enriched. Overall, this study confirmed that anaerobic digestion sludge treating nonlignocellulosic material is a potential source of microbial cellulose- and xylan-digesting functions given appropriate enrichment conditions.     

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.     

Metabolic activity of subterranean microbial communities in deep granitic groundwater supplemented with methane and H2

It was previously concluded that opposing gradients of sulphate and methane, observations of 16S ribosomal DNA sequences displaying great similarity to those of anaerobic methane-oxidizing Archaea and a peak in sulphide concentration in groundwater from a depth of 250–350 m in Olkiluoto, Finland, indicated proper conditions for methane oxidation with sulphate. In the present research, pressure-resistant, gas-tight circulating systems were constructed to enable the investigation of attached and unattached anaerobic microbial populations from a depth of 327 m in Olkiluoto under in situ pressure (2.4 MPa), diversity, dissolved gas and chemistry conditions. Three parallel flow cell cabinets were configured to allow observation of the influence on microbial metabolic activity of 11 mℳ methane, 11 mℳ methane plus 10 mℳ H₂ or 2.1 mℳ O₂ plus 7.9 mℳ N₂ (that is, air). The concentrations of these gases and of organic acids and carbon, sulphur chemistry, pH and E_h, ATP, numbers of cultivable micro-organisms, and total numbers of cells and bacteriophages were subsequently recorded under batch conditions for 105 days. The system containing H₂ and methane displayed microbial reduction of 0.7 mℳ sulphate to sulphide, whereas the system containing only methane resulted in 0.2 mℳ reduced sulphate. The system containing added air became inhibited and displayed no signs of microbial activity. Added H₂ and methane induced increasing numbers of lysogenic bacteriophages per cell. It appears likely that a microbial anaerobic methane-oxidizing process coupled to acetate formation and sulphate reduction may be ongoing in aquifers at a depth of 250–350 m in Olkiluoto.     

Phylogenetic diversity and activity of anaerobic microorganisms of high-temperature horizons of the Dagang oil field (P. R. China)

The number of microorganisms of major metabolic groups and the rates of sulfate reduction and methanogenesis processes in the formation waters of the high-temperature horizons of Dagang oil field have been determined. Using cultural methods, it was shown that the microbial community contained aerobic bacteria oxidizing crude oil, anaerobic fermentative bacteria, sulfate-reducing bacteria, and methanogens. Using cultural methods, the possibility of methane production from a mixture of hydrogen and carbon dioxide (H₂ + CO₂) and from acetate was established, and this result was confirmed by radioisotope methods involving NaH¹⁴CO₃ and ¹⁴CH₃COONa. Analysis of enrichment cultures 16S rDNA of methanogens demonstrated that these microorganisms belong to Methanothermobacter sp. (M. thermautotrophicus), which consumes hydrogen and carbon dioxide as basic substrates. The genes of acetate-utilizing bacteria were not revealed. Phylotypes of the representatives of Thermococcus spp. were found among archaeal 16S rDNA. 16S rRNA genes of bacterial clones belong to the orders Thermoanaerobacteriales (Thermoanaerobacter, Thermovenabulum, Thermacetogenium, and Coprothermobacter spp.), Thermotogales, Nitrospirales (Thermodesulfovibrio sp.) and Planctomycetales. 16S rDNA of a bacterium capable of oxidizing acetate in the course of syntrophic growth with H₂-utilizing methanogens was found in high-temperature petroleum reservoirs for the first time. These results provide further insight into the composition of microbial communities of high-temperature petroleum reservoirs, indicating that syntrophic processes play an important part in acetate degradation accompanied by methane production.     

Nutrient and acetate amendment leads to acetoclastic methane production and microbial community change in a non‐producing Australian coal well

Coal mining is responsible for 11% of total anthropogenic methane emission thereby contributing considerably to climate change. Attempts to harvest coalbed methane for energy production are challenged by relatively low methane concentrations. In this study, we investigated whether nutrient and acetate amendment of a non‐producing sub‐bituminous coal well could transform the system to a methane source. We tracked cell counts, methane production, acetate concentration and geochemical parameters for 25 months in one amended and one unamended coal well in Australia. Additionally, the microbial community was analysed with 16S rRNA gene amplicon sequencing at 17 and 25 months after amendment and complemented by metagenome sequencing at 25 months. We found that cell numbers increased rapidly from 3.0 × 10⁴ cells ml^?1 to 9.9 × 10⁷ in the first 7 months after amendment. However, acetate depletion with concomitant methane production started only after 12–19 months. The microbial community was dominated by complex organic compound degraders (Anaerolineaceae, Rhodocyclaceae and Geobacter spp.), acetoclastic methanogens (Methanothrix spp.) and fungi (Agaricomycetes). Even though the microbial community had the functional potential to convert coal to methane, we observed no indication that coal was actually converted within the time frame of the study. Our results suggest that even though nutrient and acetate amendment stimulated relevant microbial species, it is not a sustainable way to transform non‐producing coal wells into bioenergy factories.     

On the Possibility of Aerobic Methane Production by Pelagic Microbial Communities of the Laptev Sea

Microbiology - The taxonomic diversity and metabolic activity of microbial communities in the Laptev Sea water column above and outside the methane seep field were studied. The concentrations of...