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.     

Molecular biologic techniques applied to the microbial prospecting of oil and gas in the Ban 876 gas and oil field in China

Currently, molecular biologic techniques achieve a great development in studies of soil samples. The objective of this research is to improve methods for microbial prospecting of oil and gas by applying culture-independent techniques to soil sampled from above a known oil and gas field. Firstly, the community structure of soil bacteria above the Ban 876 Gas and Oil Field was analyzed based on 16S rRNA gene clone libraries. The soil bacteria communities were consistently different along the depth; however, Chloroflexi and Gemmatimonadetes were predominant and methanotrophs were minor in both bacteria libraries (DGS1 and DGS2). Secondly, the numbers of methane-oxidizing bacteria, quantified using a culture-dependent procedure and culture-independent group-specific real-time PCR (RT-PCR), respectively, were inconsistent with a quantify variance of one or two orders of magnitude. Special emphasis was given to the counting advantages of RT-PCR based on the methanotrophic pmoA gene. Finally, the diversity and distribution of methanotrophic communities in the soil samples were analyzed by constructing clone libraries of functional gene. All 508-bp inserts in clones phylogenetically belonged to the methanotrophic pmoA gene with similarities from 83% to 100%. However, most of the similarities were below 96%. Five clone libraries of methanotrophs clearly showed that the anomalous methanotrophs (Methylosinus and Methylocystis) occupy the studied area.     

Investigation of the methanotrophic communities of the hot springs of the Uzon caldera,Kamchatka, by molecular ecological techniques

The methane-oxidizing microbial communities inhabiting the bottom sediments of 36 hot springs of the Uzon caldera (Kamchatka, Russia) located in the thermal fields Vostochnoe, Oranzhevoe, and Severnoe, as well as near the lakes Fumarol’noe and Khloridnoe and the Izvilistyi stream, were studied. Methanotrophic bacteria were detected by PCR and FISH in only 8 hot springs. The highest numbers of copies of the pmoA gene (molecular marker of methanotrophy) (2.8 × 10⁷ and 1.1 × 10⁷ copies/mL sediment) were detected in the Kul’turnyi and Kvadrat springs; however, in other springs, the numbers of the pmoA gene copies were significantly lower (5.4 × 10³–2.8 × 10⁶ copies/mL sediment). By using the FISH method, only type I methanotrophs were detected in these springs, with their percentage ranging from 0.3 to 20.5% of the total number of eubacteria. PCR-DGGE analysis of the pmoA gene showed that the diversity of methanotrophs was extremely low (no more that two components). Analysis of the deduced PmoA amino acid sequences demonstrated that methanotrophic bacteria of the genus Methylothermus, closely related to representatives of two valid species, widely occurred in the thermal springs near Lake Fumarol’noe. Other bacteria differing considerably from the detected Methylothermus species were detected as well. In the springs with low pH values (2.6–3.8), methanotrophic Gammaproteobacteria most closely related to the genera Methylomonas and Methylobacter were detected for the first time.     

典型草地土壤好氧甲烷氧化的微生物生态过程

【目的】针对我国甘肃三个典型生态区草地土壤(玛曲MQ、临泽LZ和环县HX),研究其甲烷氧化潜力、甲烷氧化菌(methane-oxidizingbacteria,MOB)丰度及可能存在的群落分异规律。【方法】通过原位分析、室内高浓度甲烷模拟培养三种典型土壤及实时荧光定量、高通量测序的方法研究甲烷氧化菌标靶基因pmoA序列的组成及其丰度变化规律。【结果】三种典型草地土壤的原位甲烷氧化菌的丰度存在显著差异,表现为MQ>HX>LZ,其数量范围为为0.18–6.86×10^7g/d.w.s.;甲烷氧化潜力也表现出类似规律,其通量为109–169mg/(m^2·h);甲烷氧化潜力与原位土壤中甲烷氧化菌丰度有正相关。三种草地土壤甲烷氧化菌存在明显的空间异质性,采用高通量测序的方法,发现三种草地原位土壤中的优势类群为USCγ(Upland Soil Cluster gamma,USCγ);然而,室内高浓度甲烷氧化过程中,传统的甲烷氧化菌均发生明显增加,MQ土壤中TypeⅡ的Methylocystis为优势类群,而LZ和HX土壤的优势类群均为TypeⅠ型Methylosarcina。【结论】这些研究结果表明,我国甘肃典型草地土壤中也存在难培养的大气甲烷氧化菌和经典的可培养甲烷氧化菌,这些微生物极可能氧化极低浓度的大气甲烷,也可能利用闭蓄于土壤中的高浓度甲烷生长。未来应采用先进技术原位观测大气甲烷氧化过程并分离相应微生物类群,研究草地土壤甲烷氧化菌地理分异规律及其环境驱动机制。     

Methanotrophic community structure and activity under warming and grazing of alpine meadow on the Tibetan Plateau

Knowledge about methanotrophs and their activities is important to understand the microbial mediation of the greenhouse gas CH₄ under climate change and human activities in terrestrial ecosystems. The effects of simulated warming and sheep grazing on methanotrophic abundance, community composition, and activity were studied in an alpine meadow soil on the Tibetan Plateau. There was high abundance of methanotrophs (1.2–3.4 × 10⁸ pmoA gene copies per gram of dry weight soil) assessed by real-time PCR, and warming significantly increased the abundance regardless of grazing. A total of 64 methanotrophic operational taxonomic units (OTUs) were obtained from 1,439 clone sequences, of these OTUs; 63 OTUs (98.4%) belonged to type I methanotrophs, and only one OTU was Methylocystis of type II methanotrophs. The methanotroph community composition and diversity were not apparently affected by the treatments. Warming and grazing significantly enhanced the potential CH₄ oxidation activity. There were significantly negative correlations between methanotrophic abundance and soil moisture and between methanotrophic abundance and NH₄–N content. The study suggests that type I methanotrophs, as the dominance, may play a key role in CH₄ oxidation, and the alpine meadow has great potential to consume more CH₄ under future warmer and grazing conditions on the Tibetan Plateau.     

Activity, distribution, and abundance of methane-oxidizing bacteria in the near surface soils of onshore oil and gas fields

Methane-oxidizing bacteria (MOB) have long been used as an important biological indicator for oil and gas prospecting, but the ecological characteristics of MOB in hydrocarbon microseep systems are still poorly understood. In this study, the activity, distribution, and abundance of aerobic methanotrophic communities in the surface soils underlying an oil and gas field were investigated using biogeochemical and molecular ecological techniques. Measurements of potential methane oxidation rates and pmoA gene copy numbers showed that soils inside an oil and gas field are hot spots of methane oxidation and MOB abundance. Correspondingly, terminal restriction fragment length polymorphism analyses in combination with cloning and sequencing of pmoA genes also revealed considerable differences in the methanotrophic community composition between oil and gas fields and the surrounding soils. Principal component analysis ordination furthermore indicated a coincidence between elevated CH₄ oxidation activity and the methanotrophic community structure with type I methanotrophic Methylococcus and Methylobacter, in particular, as indicator species of oil and gas fields. Collectively, our results show that trace methane migrated from oil and gas reservoirs can considerably influence not only the quantity but also the structure of the methanotrophic community.     

Comparison of Methanotrophic Community and Methane Oxidation between Rhizospheric and Non-Rhizospheric Soils

Using particulate methane monooxygenase (pMMO) encoding gene, pmoA-based terminal-restrict fragment length polymorphism (T-RFLP), the methanotrophic communities between rhizospheric soils (RSs) and non-rhizospheric soil (NRSs) of landfill cover (LC), riparian wetland (RW) and rice paddy (RP) were compared before and after pre-incubation of 90 days. The ultimate potential of methane oxidation rate (UPMOR) and gene copy number of pmoA were evaluated in the soil samples after pre-incubation. Compared to the methanotrophic community in the soil samples before pre-incubation, type II methanotrophs, the Methylocystis-Methylosinus group, was mostly increased after pre-incubation, regardless of the soil type. The UPMOR (11.82 ± 0.27 μmol-CH₄· g^?1 _soil-DW·h^?1) in the LC-RS was significantly higher than that (9.57 ± 0.14 μmol-CH₄· g^?1 _soil-DW·h^?1) in the LC-NRS. However, no significant difference was found between RSs and NRSs in the RW (15.28 ± 0.91 and 13.23 ± 0.69 μmol-CH₄· g^?1 _soil-DW·h^?1, respectively) and RP (13.81 ± 1.04 and 12.81 ± 2.40 μmol-CH₄· g^?1 _soil-DW·h^?1, respectively) soils. There was no significantly difference in the gene copy numbers of pmoA in the RSs compared with those in the NRSs at all of the sampling sites. This study provides basic metagenomic information about both rhizospheric and non-rhizospheric methanotrophs, which will be helpful in developing a better strategy of biological methane removal from both natural and anthropogenic major methane sources.     

Methanotrophic Communities in the Soils of the Russian Northern Taiga and Subarctic Tundra

The PCR analysis of DNA extracted from soil samples taken in the Russian northern taiga and subarctic tundra showed that the DNA extracts contain genes specific to methanotrophic bacteria, i.e., the mmoX gene encoding the conserved -subunit of the hydroxylase component of soluble methane monooxygenase, the pmoA gene encoding the -subunit of particulate methane monooxygenase, and the mxaFgene encoding the -subunit of methanol dehydrogenase. PCR analysis with group-specific primers also showed that methanotrophic bacteria in the northern taiga and subarctic tundra soils are essentially represented by the type I genera Methylobacter, Methylomonas, Methylosphaera, and Methylomicrobium and that some soil samples contain type II methanotrophs close to members of the genera Methylosinus and Methylocystis. The electron microscopic examination of enrichment cultures obtained from the soil samples confirmed the presence of methanotrophic bacteria in the ecosystems studied and showed that the methanotrophs contain only small amounts of intracytoplasmic membranes.     

Molecular Existence and Diversity of Nitrite-Dependent Anaerobic Methane Oxidizing (n-Damo) Bacteria in the Lakes of Badain of the Gobi Desert

Nitrite-dependent anaerobic methane oxidation (n-damo) process, mediated by Candidatus Methylomirabilis oxyfera of the candidate phylum NC10, was discovered recently which plays an important role in coupling the global nitrogen and carbon cycles. However, the distribution and diversity of this new anaerobic methane-oxidizing microorganism have not been investigated in desert lakes yet. The present study successfully retrieved n-damo bacterial 16S rRNA and pmoA gene sequences using PCR technique from lakes in Badain Jaran Desert of China. Phylogenetic analyses showed that n-damo bacteria widely occurred in brine and freshwater lakes on the desert with high diversity, including both sediment and water samples. The results of quantitative PCR indicated that the abundance of the 16S rRNA gene in lake sediments varied from 1.12?±?0.68?×?10⁵ to 1.64?±?0.70?×?10⁵ copies g^?1 (dry weight), while that in water samples per milliliter was generally one order of magnitude lower than sediments. Correlation analyses suggested that n-damo bacterial abundance and diversity strongly depended on salinity. In lake sediments, the distribution, abundance, and diversity of n-damo bacteria were significantly associated with depth due to the concentration gradient of the NO_x^- and ammonium. This study provided new insights into both the n-damo community patterns and its interaction with ambient environmental factors in the desert lake ecosystem.     

The presence of atrazine and atrazine-degrading bacteria in the residential, cattle farming, forested and golf course regions of Lake Oconee

Aims: To assess the concentration of atrazine in Lake Oconee and develop a qPCR assay as a potential marker for the presence of atrazine‐degrading bacteria indicating atrazine contamination. Methods and Results: Water and sediment samples were collected from the Oconee Lake at four golf course sites, two residential sites, one cattle farming site and a forested site. Atrazine concentration at the study sites was determined using an ELISA kit and indicated the presence of atrazine from 0·72 ppb at the forested sites to 1·84 ppb at the golf course sites. QPCR results indicate the presence of atzA gene (atrazine chlorohydrolase) from 1·51 × 10² gene copies at the residential sites to 3·31 × 10⁵ gene copies per 100 ml of water at the golf course regions of the lake and correlated (r = 0·64) with atrazine concentration. Sediment samples had higher atzA gene copies compared with the water samples (P < 0·05). Conclusions: Atrazine concentration and the highest quantity of atzA gene were detected in the golf course regions of the lake. Overall, atrazine concentration monitored in Lake Oconee was below the Environment Protection Agency (EPA) regulatory standards. Significance and Impact of the Study: Quantitative PCR is an efficient technique for assessing the presence of atrazine catabolism gene as a functional marker for atrazine‐degrading bacteria and the presence of atrazine contamination.     

顺北典型油气藏上方土壤甲烷氧化菌菌群特征及其勘探意义

【目的】甲烷氧化细菌(MOB)长期以来一直被用作石油和天然气勘探的重要油气指示菌,其仅能利用甲烷作为唯一碳源。根据甲烷氧化菌菌群特征结合地质剖面可以较好地预测深部油气藏,为石油勘探提供良好的数据支撑。由于传统平板培养法只能针对可培养甲烷氧化菌,方法具有一定局限性。【方法】本文采用分子生物学技术结合地球化学烃类指标研究了顺北典型油气藏上方土壤中甲烷氧化菌的分布。【结果】研究结果显示,油气田上方pmoA基因拷贝数与酸解烃含量具有一定的正相关性,且油气区比背景区高0.5–2个数量级。16SrRNA基因高通量测序和pmoA基因的克隆文库结果显示顺北油藏上方土壤中甲烷氧化菌主要以I型为主,水平剖面中甲烷氧化菌随着离油田距离增加存在I型向II型演变的现象,且Methylomonassp.在背景区与油气区的丰度有较大差异,具有良好的油气指示潜力。【结论】综上所述,长期微渗透过程中甲烷氧化菌(MOB)的菌群特征对预测深层油藏具有一定的指示作用,结合地质剖面和地表烃类可以有效预测有利油藏区域。     

Diversity of Aerobic Methanotrophic Bacteria in a Permafrost Active Layer Soil of the Lena Delta,Siberia

With this study, we present first data on the diversity of aerobic methanotrophic bacteria (MOB) in an Arctic permafrost active layer soil of the Lena Delta, Siberia. Applying denaturing gradient gel electrophoresis and cloning of 16S ribosomal ribonucleic acid (rRNA) and pmoA gene fragments of active layer samples, we found a general restriction of the methanotrophic diversity to sequences closely related to the genera Methylobacter and Methylosarcina, both type I MOB. In contrast, we revealed a distinct species-level diversity. Based on phylogenetic analysis of the 16S rRNA gene, two new clusters of MOB specific for the permafrost active layer soil of this study were found. In total, 8 out of 13 operational taxonomic units detected belong to these clusters. Members of these clusters were closely related to Methylobacter psychrophilus and Methylobacter tundripaludum, both isolated from Arctic environments. A dominance of MOB closely related to M. psychrophilus and M. tundripaludum was confirmed by an additional pmoA gene analysis. We used diversity indices such as the Shannon diversity index or the Chao1 richness estimator in order to compare the MOB community near the surface and near the permafrost table. We determined a similar diversity of the MOB community in both depths and suggest that it is not influenced by the extreme physical and geochemical gradients in the active layer.     

Distribution and Diversity of Nitrite-Dependent Anaerobic Methane-Oxidising Bacteria in the Sediments of the Qiantang River

Nitrite-dependent anaerobic methane oxidation (n-damo) process was reported to be mediated by “Candidatus Methylomirabilis oxyfera”, which belongs to the candidate phylum NC10. M. oxyfera-like bacteria have been detected in lake ecosystems, while their distribution, diversity and abundance in river ecosystems have not been well studied. In this study, both the 16S rRNA and the pmoA molecular biomarkers confirmed the presence of diverse NC10 phylum bacteria related to M. oxyfera in a river ecosystem—the Qiantang River, Zhejiang Province (China). Phylogenetic analysis of 16S rRNA genes demonstrated that the recovered M. oxyfera-like sequences could be grouped into several distinct clusters that exhibited 89.8 % to 98.9 % identity to the M. oxyfera 16S rRNA gene. Similarly, several different clusters of pmoA gene sequences were observed, and these clusters displayed 85.1–95.4 % sequence identity to the pmoA gene of M. oxyfera. Quantitative PCR showed that the abundance of M. oxyfera-like bacteria varied from 1.32?±?0.16?×?10⁶ to 1.03?±?0.12?×?10⁷ copies g (dry weight)^?1. Correlation analysis demonstrated that the total inorganic nitrogen content, the ammonium content and the organic content of the sediment were important factors affecting the distribution of M. oxyfera-like bacterial groups in the examined sediments. This study demonstrated the distribution of diverse M. oxyfera-like bacteria and their correlation with environmental factors in Qiantang River sediments.     

Bacterial Community Structure in Two Permafrost Wetlands on the Tibetan Plateau and Sanjiang Plain,China

Permafrost wetlands are important methane emission sources and fragile ecosystems sensitive to climate change. Presently, there remains a lack of knowledge regarding bacterial communities, especially methanotrophs in vast areas of permafrost on the Tibetan Plateau in Northwest China and the Sanjiang Plain (SJ) in Northeast China. In this study, 16S rRNA-based quantitative PCR (qPCR) and 454 pyrosequencing were used to identify bacterial communities in soils sampled from a littoral wetland of Lake Namco on the Tibetan Plateau (NMC) and an alluvial wetland on the SJ. Additionally, methanotroph-specific primers targeting particulate methane monooxygenase subunit A gene (pmoA) were used for qPCR and pyrosequencing analysis of methanotrophic community structure in NMC soils. qPCR analysis revealed the presence of 10¹⁰ 16S rRNA gene copies per gram of wet soil in both wetlands, with 10⁸ pmoA copies per gram of wet soil in NMC. The two permafrost wetlands showed similar bacterial community compositions, which differed from those reported in other cold environments. Proteobacteria, Actinobacteria , and Chloroflexi were the most abundant phyla in both wetlands, whereas Acidobacteria was prevalent in the acidic wetland SJ only. These four phyla constituted more than 80 % of total bacterial community diversity in permafrost wetland soils, and Methylobacter of type I methanotrophs was overwhelmingly dominant in NMC soils. This study is the first major bacterial sequencing effort of permafrost in the NMC and SJ wetlands, which provides fundamental data for further studies of microbial function in extreme ecosystems under climate change scenarios.     

Improved Method for Detection of Methanotrophic Bacteria in Forest Soils by PCR

A primer set was designed for the specific detection of methanotrophic bacteria in forest soils by PCR. The primer sequences were derived from highly conservative regions of the pmoA gene, encoding the α-subunit of the particulate methane monooxygenase present in all methanotrophs. In control experiments with genomic DNA from a collection of different type I, II, and X methanotrophs, it could be demonstrated that the new primers were specific for members of the genera Methylosinus, Methylocystis, Methylomonas, Methylobacter, and Methylococcus. To test the suitability of the new primers for the detection of particulate methane monooxygenase (pMMO) containing methanotrophs in environmental samples we used DNA extracts from an acid spruce forest soil. For simple and rapid purification of the DNA extracts, the samples were separated by electrophoresis on a low-melting-point agarose gel. This allowed us to efficiently separate the DNA from coextracted humic acids. The DNA from the melted agarose gel was ready for use in PCR reactions. In PCR reactions with DNA from the Ah soil layer, products of the correct size were amplified by PCR by use of the new primers. By sequencing of cloned PCR products, it could be confirmed that the PCR products represented partial sequences with strong similarity to the pmoA gene. The sequence was most related to the pmoA sequence of a type II methanotroph strain isolated from the Ah layer of the investigated soils. Received: 1 September 2000 / Accepted: 2 October 2000     

Application of a Newly Developed ARB Software-Integrated Tool for In Silico Terminal Restriction Fragment Length Polymorphism Analysis Reveals the Dominance of a Novel pmoA Cluster in a Forest Soil

TRF-CUT, an ARB-implemented tool, was developed to predict in silico the terminal restriction fragments of aligned small-subunit rRNA gene or functional gene sequences. Application of this new tool to perform directed terminal restriction fragment length polymorphism analysis of pmoA products obtained from a forest soil revealed that novel cluster I methanotrophic bacteria were dominant.     

pmoA-Based Analysis of Methanotrophs in a Littoral Lake Sediment Reveals a Diverse and Stable Community in a Dynamic Environment

Diversity and community structure of aerobic methane-oxidizing bacteria in the littoral sediment of Lake Constance was investigated by cloning analysis and terminal restriction fragment length polymorphism (T-RFLP) fingerprinting of the pmoA gene. Phylogenetic analysis revealed a high diversity of type I and type II methanotrophs in the oxygenated uppermost centimeter of the sediment. T-RFLP profiles indicated a high similarity between the active methanotrophic community in the oxic layer and the inactive community in an anoxic sediment layer at a 10-cm depth. There were also no major changes in community structure between littoral sediment cores sampled in summer and winter. By contrast, the fingerprint patterns showed substantial differences between the methanotrophic communities of littoral and profundal sediments.     

重庆紫色水稻土中“全程”和“半程”氨氧化微生物的垂直分异

【目的】系统评估全程氨氧化细菌(complete ammonia oxidizing bacteria, Comammox bacteria)、半程氨氧化细菌(AOB)和古菌(AOA)在典型水稻土剖面的垂直分异规律。2015年发现的"全程"氨氧化细菌(Comammox Nitrospira)可将氨分子一步氧化为硝酸盐,实现硝化作用。而经典的"半程"氨氧化细菌(AOB)或古菌(AOA)将氨分子氧化为亚硝酸盐后,再由系统发育完全不同的硝化细菌将其氧化为硝酸盐。全程氨氧化细菌实现了一步硝化全过程,根本改变了学术界对2类微生物分步硝化的经典认知,但相关研究仍处于初步阶段。【方法】选择重庆北碚地区2017年典型水稻土并采集5、10、20和40 cm不同深度土壤(剖面采样点的上下误差不超过1cm),提取水稻土总DNA后,利用标靶功能基因amoA,通过实时荧光定量PCR技术分析全程氨氧化细菌(Comammox)、半程氨氧化细菌(AOB)和古菌(AOA)在水稻土不同深度的数量变异规律。【结果】半程氨氧化细菌AOB和古菌AOA均随土壤深度增加呈显著下降趋势。然而,全程氨氧化细菌的两大类微生物则表现出相反的规律,Comammox Clade A的丰度随着土壤剖面的加深而显著增加(P0.05),但Clade B并未有类似规律。Clade A在水稻土不同层次的土层中均比Clade B高出1个数量级,在5 cm和40 cm处的最低和最高值分别为3.42×10~7、8.46×10~7 copies/g。AOA与AOB的丰度大致相当,5cm剖面处数量最高分别为1.23×10~7、1.83×10~5copies/g,但其平均丰度远低于全程氨氧化细菌,Comammox与AOA、AOB amoA功能基因拷贝数之比为10–2000。【结论】全程氨氧化细菌(Comammox bacteria)广泛分布于水稻土不同土层中,且数量远高于"半程"氨氧化细菌和古菌,意味着Comammox可能在水稻土硝化作用中起重要作用。