气态烃诱导下油气微生物数量及功能基因变化特征

【目的】烃类渗漏是地球化学循环中的自然垂直运移现象。油气微生物勘探技术就是通过检测油气微生物在烃类渗漏条件下形成的异常特征进而预测下伏油气藏。然而,上浮烃类比重小,使得油气微生物丰度小,缺乏对油气藏地表烃类与油气微生物的深入认识。【方法】本研究在人工模拟环境下,采用培养法和定量PCR对油气微生物数量和油气功能基因的变化特征进行研究。【结果】人工模拟的不同渗漏环境,在一定的驯化培养周期下分别考察甲烷氧化菌和丁烷氧化菌数量变化特征,甲烷氧化菌与丁烷氧化菌在不同烃源中呈现不同的发育情况;同时,在气态烃高浓度阳性对照和微渗漏条件下,油气指示基因pmo A与bom X基因丰度呈现增长现象,然而经过吹脱实验基因丰度仍能指示出曾经发育的油气微生物,在油气微生物勘探检测分析时间尺度上精度高于数量水平的检测。【结论】本研究考察了土壤中油气微生物的数量和功能基因变化特征,为烃类渗漏与油气微生物之间的相关性研究奠定了基础,为油气微生物勘探提供直接实验依据。     

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。     

一种基于BIOLOG代谢指纹分析的油气微生物勘探新方法

【目的】建立基于土壤微生物群落生理功能代谢的油气微生物勘探新方法。【方法】采集新疆玛北油气藏区浅层地表土壤样品45份,非油气藏区样品25份,利用BIOLOG微平板,测定微生物群落代谢活性,采用判别分析模型判断油气藏区微生物异常。【结果】从95种碳源中,筛选到10种能够体现油气藏区与非油气藏区土壤微生物群落结构差异的典型碳源,利用判别函数对试验及对照区进行回判,油气藏区判别正确率为97.8%,非油藏区判别正确率为100%,总判别正确率为98.6%。【结论】BIOLOG技术能高效准确的用于油气藏初步勘测。     

油气田土壤甲烷氧化菌实时荧光定量PCR检测技术的 建立与应用

【目的】建立快速检测甲烷氧化菌含量的SYBR GreenⅠ实时荧光定量PCR技术,用于油气微生物勘探。【方法】以含有甲烷氧化菌功能基因pmoA片段的重组质粒为标准品,优化实验条件,建立标准曲线,进行敏感性、重复性和特异性评价,并将该技术用于实际样品的检测。【结果】该技术标准曲线的相关系数R2为0.999 9,扩增效率为99.976%,检测范围为3.897×101-3.897×109 copies/μL,检出限约为40 copies/μL,重复性实验中CT值的变异系数优于3%,对12种非甲烷氧化菌均没有扩增,显示该技术具有很好的敏感性、重复性和特异性。利用该技术对气田、油田和非油气田土样进行了检测,发现气田具有明显的异常高值。【结论】为油气田的勘探建立了一种高效、特异、灵敏、准确的甲烷氧化菌荧光定量PCR检测技术,同时为其它指示菌检测技术的建立提供了参考。     

甲烷氧化菌在勘测油气藏方面的应用

甲烷氧化菌是可用于勘测石油气藏的一种重要的微生物。探究得到不同区块的石油气藏上方土壤中微生物的构成与群落数量之间的差别及关系,可为地球化学地表石油气勘探等工作提供一些技术支持和理论基础。对甲烷氧化菌群落结构的不同与石油气藏上方土壤之间关系进行研究分析,从而得出石油气藏上方土壤中甲烷氧化菌的生理生化反应及其群落结构特点,可为石油气藏的下伏地理位置指明范围,为石油气藏的微生物勘探提供理论基础。     

石油微生物学在中国科学院微生物研究所的发展

本文较详尽地综述了中国科学院微生物研究所50多年在涉及石油微生物学的科学研究成果,包括微生物勘探法和气态烃氧化菌、微生物与油气形成、有机酸的生产、油田微生物与提高采油量、微生物提高采油的现场应用.共引用了105篇文献.     

风干土壤中氨氧化微生物的恢复

【目的】比较历史风干土壤与加水恢复培养土壤中氨氧化古菌AOA和细菌AOB的组成与数量差异,探究风干土壤用于后续微生物生理生态学研究的可能性;明确我国典型酸性森林土壤中,海洋类Group 1.1a是否为数量上占据优势的古菌AOA生态型。【方法】针对中国生态系统研究网络10个台站的典型森林土壤样品,围绕风干保存和加水培养两种处理,通过高通量测序土壤氨氧化古菌及细菌amoA标靶基因,分析氨氧化微生物群落组成的变化规律;利用实时荧光定量PCR和DGGE指纹图谱技术,研究森林土壤微生物群落16S rRNA基因的数量变化规律,以及氨氧化细菌和古菌群落结构的差异。【结果】10个历史风干土壤加水培养28天后,土壤细菌和古菌数量均急剧增加,最高可达3230倍和568倍;其中8个土壤中氨氧化古菌AOA明显增加,5个土壤中氨氧化细菌AOB表现出明显的增加趋势。然而,高通量测序和系统发育分析表明,历史风干土壤与加水恢复培养土壤中AOA和AOB的群落组成无明显变化。Group 1.1b是氨氧化古菌的优势类群,而氨氧化细菌的主要类群是Nitrosospira螺菌属。氨氧化古菌和细菌的比例与总氮浓度呈显著正相关(r2=0.54,P0.05),表明酸性条件下土壤矿化并提供铵态氮底物可能是古菌氨氧化的驱动机制。【结论】风干土壤加水恢复培养后,AOA和AOB的种群数量大多出现增加的趋势,但其物种组成未发生显著变化,表明风干保存的土壤样品可用于后续室内培养,开展微生物生理生态学研究。与已有的海洋AOA生态型主导酸性土壤氨氧化类群的报道不同,土壤Group 1.1b是本研究森林土壤中的优势类群。     

温州市境内城市河流底泥氨氧化菌富集培养物微生物群落结构分析

【背景】城市河流底泥含有丰富的微生物资源,底泥表面更是硝化作用的主要位点之一,其表面微生物在河流生态系统氮的转化过程中发挥着重要作用。【目的】以温州市境内的城市河流水系温瑞塘河茶山段舜岙河和横江河的4条河道作为采样点,比较分析4种不同环境下城市河流表层底泥氨氧化菌富集培养物的微生物群落结构。【方法】通过野外采样及室内培养对底泥中氨氧化功能菌进行富集培养,采用高通量测序技术分析微生物群落的组成、丰度和多样性。【结果】富集培养后主要优势类群为变形菌门(Proteobacteria)和拟杆菌门(Bacteroidetes)。4个样品共涉及氨氧化细菌3个属,分别为亚硝化单胞菌属(Nitrosomonas)、亚硝化螺菌属(Nitrosospira)、亚硝化球菌属(Nitrosococcus),涉及氨氧化古菌1个属为Nitrososphaera,其中所有样品均以Nitrosomonas为主。不同底泥富集样品氨氧化微生物可操作分类单元(Operational taxonomic unit,OTU)组成存在明显差异,栽种有水生植物的河道底泥样品DA2具有最高的氨氧化细菌OTU数量和相对丰度,而存在生活餐饮污染的河道底泥样品DA4具有最高的氨氧化古菌OTU数量和相对丰度;相较于滞留水体,采自相对流动水体的富集样品DA2、DA4具有更高的氨氧化微生物OTU数量和相对丰度。【结论】阐述了4种不同环境下城市河流底泥氨氧化菌富集培养物微生物群落结构的多样性,确定了富集培养之后的优势类群,为氨氧化微生物培养源的选择提供了参考,也为城市河流底泥中氨氧化菌进一步的筛选分离及其生理生态特征的研究提供了科学依据。     

内源微生物驱油及其对油藏微生物活动的影响

【目的】新疆油田六中区为典型水驱普通稠油油藏，水驱效果较差，油藏具有丰富的内源微生物，本研究通过分析内源微生物驱油对油藏微生物活动的影响，确定内源微生物驱油技术在该类油藏的应用潜力。【方法】采用高通量测序及分析化学技术，系统研究实施内源微生物驱油技术后油藏细菌群落结构组成、细菌总数和功能菌群的浓度以及采出液的流体性质，总结内源微生物驱油对油藏微生物活动的影响。【结果】现场试验注入激活剂和空气后，内源微生物被显著激活，细菌群落结构发生明显变化，细菌总数及功能菌群浓度普遍提高了2–3个数量级；各种内源微生物代谢活动显著增强，与地层流体相互作用后，原油明显被乳化，最终石油采收率提高5.2%。【结论】对于内源微生物较为丰富的水驱普通稠油油藏，内源微生物驱油技术对油藏微生物活动的影响显著，具有显著的技术优势和较大的应用潜力，微生物群落结构、功能菌群浓度及其相关代谢产物可以作为评价内源微生物驱油现场激活效果的重要指标，为其他内源微生物驱油现场试验提供技术参考。     

不同成熟度煤样产甲烷潜力

摘要:【目的】评估不同类型煤炭生物降解转化为甲烷的潜力,研究原位煤层的微生物群落结构特征。【方法】分别在原位模拟、补加烃降解产甲烷菌系和补加碳源下厌氧培养煤样,利用气相色谱监测甲烷产生趋势,及高通量测序技术研究原位煤层的细菌和古菌群落。【结果】10个样品中有3个高成熟度煤样可以被厌氧降解转化为甲烷。通过生物强化和添加外源底物可以促进HF煤样的产甲烷潜力。其中SL 煤样中的古菌类群主要是氢营养型产甲烷菌Methanoculleus和乙酸营养型产甲烷菌Methanosaeta为主,细菌类群主要 属于Firmicutes(54.4%)、Proteobacteria(30.9%)、未培养微生物(10.8%)、Caldiserica(1.5%)及Thermotogae(1.3%)。【结论】不同成熟度煤样降解产气潜力不同,在部分原位煤层中可能存在参与烃降解与甲烷产生的功能菌。     

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.     

Responses of Microbial Communities to Light-Hydrocarbon Microseepage and Novel Indicators for Microbial Prospecting of Oil/Gas in the Beihanzhuang Oilfield,Northern Jiangsu,China

Previous studies on microbial prospecting of oil/gas only focused on the anomalies of light hydrocarbon-oxidizing microbes as main exploratory indicators and their exploration applications. In this study, we investigated the responses of microbial communities to light-hydrocarbon microseepage in the Beihanzhuang Oilfield, eastern China using denaturing gradient gel electrophoresis (DGGE) analysis and by comparing the difference of two-type areas with high- and low-flux light-hydrocarbon seepages. The results showed that the high-flux light-hydrocarbon seepage favored the growth of Nocardioides, Aciditerrimonas, sulphate-reducing bacteria (SRBs) related to Desulfosporosinus and Desulfovibrio, and Chloroflexi bacteria (b-7), implying that their anomalies might be adopted as novel subsidiary indicators for microbial prospecting of oil/gas in the Beihanzhuang Oilfield. Based on the newly obtained results, we have proposed a general strategy for microbial prospecting of oil/gas, i.e., to determine the anomalies of light hydrocarbon-oxidizing microbes, to select subsidiary indicators for microbial prospecting of oil/gas based on an assessment of the responses of microbial communities to light-hydrocarbon microseepage, to quantitatively measure subsidiary indicators and delimit their anomalies, to comprehensively interpret all microbial anomalies, and to make a suggestion for oil/gas prospecting. This general strategy with novel indicators may provide a more comprehensive evaluation for light-hydrocarbon microseepage and the corresponding anomalies, thereby reducing the exploration risk of oil/gas.     

Heterotrophic Potentials and Hydrocarbon Biodegradation Potentials of Sediment Microorganisms Within the Athabasca Oil Sands Deposit

Techniques for the enumeration and the determination of the potential activity of disturbed sediment mixed populations at control sites and sites within the Athabasca oil sands formation were applied to August and December samples. These techniques included the determination of general heterotrophic potential for the assimilation and respiration of glutamate, which indicated no oil sand-related changes in the sediments but which indicated a significant seasonal change. Enumeration by epifluorescence direct counts, oil sand hydrocarbon plate counts, and most-probable-number determinations of [¹⁴C]hexadecane and [¹⁴C]-naphthalene degraders indicated that only the plate count was sensitive to increased numbers of oil sand-related hydrocarbon-oxidizing microorganisms within the oil sands deposit. Unlike the most probable number determinations of [¹⁴C]hexadecane and [¹⁴C]naphthalene degraders, however, the biodegradation potential results of these substrates indicated a significant increase in activity at oil sands sites. These biodegradation potentials also showed a marked seasonal fluctuation. Although the biodegradation potentials and the endogenous hydrocarbon plate counts indicated an oil sand-adapted mixed sediment population, the results of these techniques did not correlate well with the concentrations of bituminous hydrocarbons in the sediments. The results suggest that a general capability for hydrocarbon oxidation exists in the Athabasca River system and that this capability is enhanced within the natural bounds of the Athabasca oil sands.     

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.     

Microbial processes in the Athabasca Oil Sands and their potential applications in microbial enhanced oil recovery

The Athabasca Oil Sands are located within the Western Canadian Sedimentary Basin, which covers over 140,200 km² of land in Alberta, Canada. The oil sands provide a unique environment for bacteria as a result of the stressors of low water availability and high hydrocarbon concentrations. Understanding the mechanisms bacteria use to tolerate these stresses may aid in our understanding of how hydrocarbon degradation has occurred over geological time, and how these processes and related tolerance mechanisms may be used in biotechnology applications such as microbial enhanced oil recovery (MEOR). The majority of research has focused on microbiology processes in oil reservoirs and oilfields; as such there is a paucity of information specific to oil sands. By studying microbial processes in oil sands there is the potential to use microbes in MEOR applications. This article reviews the microbiology of the Athabasca Oil Sands and the mechanisms bacteria use to tolerate low water and high hydrocarbon availability in oil reservoirs and oilfields, and potential applications in MEOR.     

高通量测序分析多种典型生境中厌氧氨氧化细菌的多样性分布特征

【背景】厌氧氨氧化过程是氮素循环过程的重要途径之一,在氮素循环中发挥重要作用。先前的研究已经证实了厌氧氨氧化细菌存在于多种生境中,但对其多样性分布还没有系统的研究。【目的】对厌氧氨氧化细菌在不同类型生境中的多样性分布规律进行深入分析,充分展示其在不同生境中的群落结构特点,并揭示多样性分布与环境因素之间的关系。【方法】在建立厌氧氨氧化细菌16S rRNA基因序列数据库的基础上,运用高通量测序技术分析其在不同生境中的多样性分布特征。【结果】厌氧氨氧化细菌在红树林、海湾和河口生境中的多样性水平较高,而污泥和红壤的多样性水平明显较低。系统发育分析表明,这些生境中的厌氧氨氧化细菌主要由Candidatus Brocadia、Ca.Scalindua和未明确分类地位的菌属组成;从河流到红树林生态系统,随着盐度的增加,厌氧氨氧化细菌的优势种属由Ca. Brocadia转变到Ca. Scalindua,相关性分析也表明了盐度是导致不同生境中厌氧氨氧化细菌群落结构差异的主要因素。【结论】不同生境中存在不同的厌氧氨氧化细菌种群结构,环境条件的差异影响了厌氧氨氧化细菌的种群分布和系统演化。     

超低渗油藏微生物吞吐技术的矿场试验

【目的】通过对渭北低渗油藏内源微生物的研究,考察分离纯化的内源解烃菌产生表面活性剂和降解原油的能力、岩心驱替增油效率,同时验证其在超低渗油田单井吞吐矿场实验的应用效果,探讨微生物采油技术在超低渗油田提高采收率的工艺和可行性。【方法】采集超低渗油藏的油水样,应用油平板进行产表面活性剂解烃菌的分离,通过生理生化特性和16S r RNA基因序列分析对菌株进行种属鉴定,评价其油藏环境适应性,利用内源-外源功能微生物复配体系进行原油降解,在填砂管和岩心物模上进行驱油实验,将优化好的微生物复配体系应用于现场实施单井吞吐工艺的实验。【结果】从渭北某区块超低渗油藏的原油样品中分离得到一株铜绿假单胞菌(Pseudomonas aeruginosa),命名为WB-001。该菌株可使发酵液的表面张力降至29.04 m N/m,使渭北原油蜡质含量降至8.48%。填砂管实验表明WB-001与外源枯草芽胞杆菌OPUS-HOB-001(Bacillus subtilis)复配后,驱油效率较单纯水驱提高了9.72%;岩心驱替实验较水驱提高12.54%。微生物单井吞吐措施后,平均日产油由措施前的0.42 t增加到0.89 t,累计增油44.47 t;原油降粘率为11.70%,降凝率为9.41%,采出水表面张力降低幅度为18.93%。【结论】通过详细的室内评估和成功的矿场实验,证明微生物采油技术在超低渗油藏有一定的应用可行性,并为后续规模化应用提供了理论基础和物质基础,为超低渗油田的高效精细开发探索一条新的途径。