A DNA-based Analysis of a Microbial Technique for the Prospecting of Oil and Gas Applied to a Known Oil Field,China

To evaluate the application of a DNA-based analysis of a microbial technique for oil and gas prospecting, the hydrocarbon potential predicted using the methanotrophic pmoA and propanotrophic prmA genes as targets was investigated in soil above a known oil reservoir. In total, 82 soil samples were collected at 50-cm depths above the Xiliu oil reservoir for quantitative real-time polymerase chain reaction (RT-PCR) analysis. The pmoA gene content ranged from 0 to 2.7 × 10⁶ copies/g dw, whereas that of prmA varied from 0 to 1.6 × 10⁶ copies/g dw. Microbial anomaly distribution maps of normalized values for the prmA gene as a main indicator suggested that the area with the highest potential of prospecting was an undeveloped location, and an area with a relatively large prmA background was identified at this oil-producing site. The areas of microbial anomaly exhibited different prmA/pmoA ratios (20, 1–5, 0.0n–0.25 and 0), and this ratio may be used as an indicator for predicting the properties of oil and gas reservoirs and biogenic methane or the influence of oil-producing activities. Our results suggest that a DNA-based analysis of a microbial technique is a powerful tool for oil and gas prospecting. This study provides a new microbial technique for the prospecting of oil and gas.     

Oxidative mitigation of aquatic methane emissions in large Amazonian rivers

The flux of methane (CH₄) from inland waters to the atmosphere has a profound impact on global atmospheric greenhouse gas (GHG) levels, and yet, strikingly little is known about the dynamics controlling sources and sinks of CH₄ in the aquatic setting. Here, we examine the cycling and flux of CH₄ in six large rivers in the Amazon basin, including the Amazon River. Based on stable isotopic mass balances of CH₄, inputs and outputs to the water column were estimated. We determined that ecosystem methane oxidation (MOX) reduced the diffusive flux of CH₄ by approximately 28–96% and varied depending on hydrologic regime and general geochemical characteristics of tributaries of the Amazon River. For example, the relative amount of MOX was maximal during high water in black and white water rivers and minimal in clear water rivers during low water. The abundance of genetic markers for methane‐oxidizing bacteria (pmoA) was positively correlated with enhanced signals of oxidation, providing independent support for the detected MOX patterns. The results indicate that MOX in large Amazonian rivers can consume from 0.45 to 2.07 Tg CH₄ yr^?1, representing up to 7% of the estimated global soil sink. Nevertheless, climate change and changes in hydrology, for example, due to construction of dams, can alter this balance, influencing CH₄ emissions to atmosphere.     

水位埋深和微地貌对金川泥炭土壤微生物活性及甲烷功能基因的影响

泥炭沼泽是长期储存碳最有效的陆地生态系统.水文特征和微地貌可能会通过调控微生物群落和功能影响泥炭地碳储存.本研究以长白山金川泥炭沼泽为研究对象,选取-10、-1、0、4、10、13、14和18 cm八个水位埋深,并在各水位埋深点采集臌囊薹草(Carexs chmidtii)草丘和丘间微地貌的土壤样品,以探究水位埋深和微...     

Diversity of functional genes for methanotrophs in sediments associated with gas hydrates and hydrocarbon seeps in the Gulf of Mexico

Methanotrophs are ubiquitous in soil, fresh water and the open ocean, but have not been well characterized in deep-sea hydrocarbon seeps and gas hydrates, where methane is unusually abundant. Here we report the presence of new functional genes for the aerobic oxidation of methane by methanotrophs in marine sediments associated with gas hydrates and hydrocarbon seeps in the Gulf of Mexico. Samples were collected from two hydrate locations (GC185 and GC234): one hydrocarbon-seep location at a brine pool (GC233) and one background-marine location about 1.2 miles north of the brine pool (NBP). Community DNA was extracted from each location to establish clone libraries for the pmoA functional gene using a PCR-based cloning approach. Three hundred and ninety clones were screened by sequencing and 46 operational taxonomic units were obtained. Eight operational taxonomic units were present in every sample; one of them was predominant and accounted for 22.8-25.3% of each clone library. Principal-component analysis indicated that samples GC185 and GC234 were closely related and, along with GC233, were significantly different from NBP. These results indicate that methanotrophic communities may be similarly impacted by hydrocarbons at the gas-hydrate and seep sites, and can be distinguished from methanotrophic communities in the normal marine sediment. Furthermore, cluster analysis showed that 84.8% of operational taxonomic units from all samples formed distinct clusters, which could not be grouped with any published pmoA sequences, indicating that a considerable number of novel methanotrophic species may exist in the Gulf of Mexico.     

Abundance and Diversity of Methanotrophs and Propanotrophs in Soils above Yangxin Oil Reservoir,China

Methanotrophs have long been used as an important biological indicator for prospecting of oil and gas, while the indication of propanotrophs in hydrocarbon micro-seep systems is still poorly investigated. In this study, the abundance and diversity of the methanotrophic pmoA gene and the propanotrophic prmA gene as target genes were investigated in soils above Yangxin oil reservoir and Beiguan non-petroliferous area using molecular biological techniques. A total of 14 soil samples were collected at different depths (5, 20, 50, 100, 150, 200 and 250 cm) of two 2.5-m soil profiles located separately within the oil field and the non-petroliferous area for analysis of fluorescent quantitative real-time polymerase chain reaction (RT-PCR) (14 samples) and clone libraries (4 samples). The results demonstrated high presence of the propanotrophic prmA gene ranging from 7.68 × 10⁵ to 2.29 × 10⁷ copies/g dw (gene copies per gram soil of dry weight) in soil from the oil field relative to the non-petroliferous area for which the same measurements yielded results all below detection limit except for the 5-cm sample. On the other hand, oil field soil yielded much lower content of the methanotrophic pmoA gene (below detection limit to 5.6 × 10² copies/g dw) than the non-petroliferous area (1.14 × 10³ copies/g dw to 1.26 × 10⁵ copies/g dw) below 20-cm depth due to influence of biogenic methane, implying that propanotrophs may be better indicator bacteria for prospecting of oil and gas. Almost all pmoA clones of two 50-cm soil samples phylogenetically belonged to Gamma-Proteobacteria and the predominant pmoA OTUs were all uncultured bacteria. All prmA clones of two 5-cm soil samples were derived predominantly from Actinobacteridae (25.7%) and Alpha-Proteobacteria (74.3%), and all dominant prmA OTUs were also clustered with uncultured bacteria. Our results confirm that propanotrophs may be better indicator bacteria for prospecting of oil and gas and enrich the knowledge on diversity of methanotrophs and propanotrophs in the oil field and the non-petroliferous area.     

Microbial diversity in sediments associated with a shallow methane seep in the tropical Timor Sea of Australia reveals a novel aerobic methanotroph diversity

This study examined the diversity of Bacteria, Archaea and in particular aerobic methanotrophs associated with a shallow (84 m) methane seep in the tropical Timor Sea, Australia. Seepage of thermogenic methane was associated with a large carbonate hardground covered in coarse carbonate-rich sediments and various benthic organisms such as solitary corals. The diversity of Bacteria and Archaea was studied by analysis of cloned 16S rRNA genes, while aerobic methanotrophic bacteria were quantified using real-time PCR targeting the α-subunit of particulate methane monooxygenase ( pmoA ) genes and diversity was studied by analysis of cloned pmoA genes. Phylogenetic analysis of bacterial and archaeal 16S rRNA genes revealed diverse and mostly novel phylotypes related to sequences previously recovered from marine sediments. A small number of bacterial 16S rRNA gene sequences were related to aerobic methanotrophs distantly related to the genera Methylococcus and Methylocaldum . Real-time PCR targeting pmoA genes showed that the highest numbers of methanotrophs were present in surface sediments associated with the seep area. Phylogenetic analysis of pmoA sequences revealed that all phylotypes were novel and fell into two large clusters comprised of only marine sequences distantly related to the genera Methylococcus and Methylocaldum that were clearly divergent from terrestrial phylotypes. This study provides evidence for the existence of a novel microbial diversity and diverse aerobic methanotrophs that appear to constitute marine specialized lineages.     

Microbial mechanism for rice variety control on methane emission from rice field soil

Rice variety is one of the key factors regulating methane (CH₄) production and emission from the paddy fields. However, the relationships between rice varieties and populations of microorganisms involved in CH₄ dynamics are poorly understood. Here we investigated CH₄ dynamics and the composition and abundance of CH₄‐producing archaea and CH₄‐oxidizing bacteria in a Chinese rice field soil planted with three types of rice. Hybrid rice produced 50–60% more of shoot biomass than Indica and Japonica cultivars. However, the emission rate of CH₄ was similar to Japonica and lower than Indica. Furthermore, the dissolved CH₄ concentration in the rhizosphere of hybrid rice was markedly lower than Indica and Japonica cultivars. The rhizosphere soil of hybrid rice showed a similar CH₄ production potential but a higher CH₄ oxidation potential compared with the conventional varieties. Terminal restriction fragment length polymorphism analysis of the archaeal 16S rRNA genes showed that the hydrogenotrophic methanogens dominated in the rhizosphere whereas acetoclastic methanogens mainly inhabited the bulk soil. The abundance of total archaea as determined by quantitative (real‐time) PCR increased in the later stage of rice growth. However, rice variety did not significantly influence the structure and abundance of methanogenic archaea. The analysis of pmoA gene fragments (encoding the α‐subunit of particulate methane monooxygenase) revealed that rice variety also did not influence the structure of methanotrophic proteobacteria, though variable effects of soil layer and sampling time were observed. However, the total copy number of pmoA genes in the rhizosphere of hybrid rice was approximately one order of magnitude greater than the two conventional cultivars. The results suggest that hybrid rice stimulates the growth of methanotrophs in the rice rhizosphere, and hence enhances CH₄ oxidation which attenuates CH₄ emissions from the paddy soil. Hybrid rice is becoming more and more popular in Asian countries. The present study demonstrated that planting of hybrid rice will not enhance CH₄ emissions albeit a higher grain production than the conventional varieties.     

Existence of Novel Phylotypes of Nitrite-Dependent Anaerobic Methane-Oxidizing Bacteria in Surface and Subsurface Sediments of the South China Sea

Nitrite-dependent anaerobic methane oxidation (n-damo) is a recently discovered new microbial process performed by the Candidatus Methylomirabilis oxyfera with an unusual intra-aerobic pathway, but there is no report about n-damo bacteria in marine environments. M. oxyfera-like sequences were successfully retrieved for the first time from both surface and subsurface ocean sediments of the South China Sea (SCS) using both 16S rRNA and pmoA genes as biomarkers and PCR amplification in this study. The majority of M. oxyfera-like 16S rRNA gene-based PCR amplified sequences from the SCS sediments formed a new group distinctively different from those detected in freshwater habitats and the information is consistent phylogenetically with those obtained from the pmoA gene. This study showed the existence of n-damo in ocean sediments and suggests that marine sediments harbor n-damo phylotypes different from those in the freshwater. This finding here expands our understanding on the distribution of n-damo bacteria to marine ecosystem and implies their potential contribution to the marine C and N cycling.