大庆油田油藏采出水的细菌群落结构

采用ARDRA (扩增性rDNA限制性酶切片段多态性分析)技术对大庆油田聚驱、水驱和过渡带3种油藏采出水中的细菌群落的基因组总DNA的16S rDNA克隆文库进行分析,研究了细菌群落结构.结果表明：随机挑取的596个阳性克隆可分为85个操作分类单元 (OTUs),其中聚驱、水驱和过渡带文库分别含有28、41和33个.通过对优势OTUs测序,并与GenBank进行序列比对,发现油藏采出水中的优势菌群为不动杆菌属、弓形杆菌属、厚壁菌门、假单胞菌属和硫磺单胞菌属.聚驱样品中细菌群落组成最简单,优势菌群为不动杆菌属,占库容的85%,假单胞菌属占7%;水驱样品中的优势菌群也是不动杆菌属,占库容的62%,假单胞菌属和硫磺单胞菌属各占20%和6%;过渡带文库的细菌群落的优势菌群为弓形杆菌属,占库容的50%,不动杆菌属和厚壁菌门各占19%和18%.     

云南省蒙自酸性矿山排水微生物群落结构和功能

为探究酸性矿山排水生态系统不同环境中的微生物群落和功能,全面了解酸性矿山排水的形成和发展规律,采用高通量测序技术研究云南省蒙自某矿区酸矿水坑和周边溪水中的原核微生物群落组成,并结合样本理化特征分析影响群落结构的主要因素,进而解析菌群的环境功能。研究发现酸矿水坑中主要有广古菌门、变形菌门(包括α、γ和δ变形菌纲)、硝化螺菌门、厚壁菌门、放线菌门和酸杆菌门等类群,与周边溪水的群落结构具有明显差异。群落多样性与pH呈显著正相关,而热原体纲(Thermoplasmata)与pH呈负相关,可对群落结构起主导作用。酸矿水坑不同样本中均具有高丰度的亚铁原体属Ferroplasma (6.60%–86.34%),酸硫杆菌属Acidithiobacillus是酸矿水和沉积泥中主要的铁、硫氧化细菌,而专性铁氧化的钩端螺旋菌属Leptospirillum的丰度较低,铁卵形菌属Ferrovum几乎只存在于酸矿水中;此外,嗜酸或耐酸的异养菌广泛分布于酸矿水和沉积泥中,它们可促进铁、硫氧化菌的生长及催化矿石溶解。结果表明,pH通过影响微生物多样性和菌群分布而对酸性矿山排水环境微生物群落结构造成重大影响。     

好氧-厌氧混合污泥启动微生物燃料电池产电性能及微生物群落动态特征

【目的】为探讨好氧-厌氧混合污泥启动微生物燃料电池(Microbial fuel cell,MFC)产电性能以及MFC对微生物群落的选择作用,【方法】以乳酸为底物,应用不依赖于培养的微生物分子生物学技术解析单室MFC启动过程中微生物群落的组成和结构动态学特征。【结果】结果表明,MFC经过3个周期启动成功,最高输出电压230 m V。当MFC外电阻为1656Ω时,最大功率密度11.15 W/m3,电池运行稳定。混合污泥启动MFC以后,阳极生物膜微生物群落结构同种泥差异较大,且多样性降低。生物膜中微生物类群按丰度依次为β-变形菌纲(Betaproteobacteria)24.90%、拟杆菌门(Bacteroidetes)21.30%、厚壁菌门(Firmicutes)9.70%、γ-变形菌纲(Gammaproteobacteria)8.50%、δ-变形菌纲(Deltaproteobacteria)7.90%、绿弯菌门(Chloroflexi)4.20%以及α-变形菌纲(Alphaproteobacteria)3.60%。有利于生物膜形成与稳定的动胶菌属(Zoogloea)和不动杆菌属(Acinetobacter)序列丰度分别占生物膜群落的5.00%和3.90%,与MFC产电能力直接相关的地杆菌属(Geobacter)序列由混合污泥中的0.60%上升至阳极生物膜中的2.60%。【结论】本研究表明,MFC阳极生物膜在驯化过程中对污泥中的微生物进行淘汰和选择,最终驯化形成了有利于生物膜形成与稳定、有机物厌氧发酵与产电的微生物菌群。     

不同激活剂对油藏内源微生物群落结构的影响

目的筛选大庆油田聚驱后油藏内源微生物采油激活剂的类型。方法在动态物理模拟实验模型基础上,利用基于16S rDNA的基因克隆文库方法,分析了两组激活剂配方激活的微生物群落结构。结果配方1激活的微生物菌群主要有假单胞菌属(75%)、深海弯曲菌属(18%)、陶厄菌属(6%);配方2激活的微生物菌群主要有陶厄菌属(50%)、假单胞菌属(26%)、螺杆菌属(15%)、梭菌属(9%)。结论激活剂配方2更适合大庆油田聚驱后油藏内源微生物采油。     

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。     

基于高通量测序的4种硝化细菌富集培养物微生物群落结构分析

【目的】比较分析4种硝化细菌富集培养物(以铵盐为氮源的淡水富集物A、以亚硝酸盐为氮源的淡水富集物B、以铵盐为氮源的低温淡水富集物C和以亚硝酸盐为氮源的海水富集物D)的微生物群落结构与组成。【方法】分别提取样品的总DNA,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】在不同微生物的分类水平,4个样品共检测到24门47纲129属。4个样品的优势菌门均为变形菌门;样品A、B、C的优势菌纲为β-变形菌纲和γ-变形菌纲,样品D的优势菌纲为γ-变形菌纲、δ-变形菌纲和芽孢杆菌纲;而优势菌属各不相同,其中样品A为亚硝化单胞菌属(24.56%),样品B为链霉菌属(7.15%),样品C为噬菌弧菌属(19.36%)和类诺卡氏菌属(19.35%),样品D为嗜酸菌属(13.6%)和柄杆菌属(11.5%)。共检测出7种具有硝化功能的细菌,其中样品A、B和D中主要是亚硝化单胞菌属,占比分别为24.56%、4.94%和0.63%,样品C主要为Nitrospirillum(0.69%)和硝化螺旋菌属(0.69%)。此外在样品中还检测到红灯食烷菌、羽扇豆根瘤菌等有益菌,以及弧菌属、伯克霍尔德菌等致病菌。【结论】阐述了4个样品微生物群落结构的多样性,确定了不同培养物中起主要作用的硝化细菌类群以及其它与环境物质循环相关或具有特殊生理特性的菌群,研究结果为硝化细菌富集培养物的实际应用奠定了基础。     

系统性红斑狼疮小鼠肠道微生物与抗dsDNA抗体水平的相关性

目的探讨系统性红斑狼疮(简称狼疮)TC小鼠肠道微生物菌群结构与抗ds DNA抗体之间的关系。方法 ELISA法检测狼疮TC小鼠血清抗ds DNA抗体,然后分别收集ds DNA阳性组和阴性组的小鼠粪便,利用Illumina Hi Seq 2500高通量测序,进行粪便样本16S测序,比较两组之间的肠道微生物菌群结构与抗ds DNA抗体水平之间的关系。结果通过ELISA检测抗体结果和小鼠粪便高通量测序结果显示,ds DNA阳性组TC小鼠的物种群落多样性显著低于ds DNA阴性组;同时,两组TC小鼠肠道微生物分别在门水平Chloroflexi(绿弯菌门);纲水平Betaproteobacteria(β变形菌纲)、Deltaproteobacteria(δ变形菌纲)和Ktedonobacteria(纤线杆菌纲);目水平Burkholderiales(伯克氏菌目)、Desulfovibrionales(脱硫弧菌目)和Ktedonobacterales(纤线杆菌目);科水平Alcaligenaceae(产碱菌科)和Desulfovibrionaceae(脱硫弧菌科);属水平Parasutterella(副萨特氏菌属)和Desulfovibrio(脱硫弧菌属)上差异有显著性(P0.05)。结论狼疮TC小鼠肠道微生物菌群结构与抗ds DNA抗体高度相关,本研究为进一步阐明肠道微生物菌群与系统性红斑狼疮发病机制的关系提供依据。     

T-RFLP技术分析油藏微生物多样性

应用T-RFLP(末端限制性片段长度多态性)技术分析和比较了胜利油田单12区块的一口注水井(S12-zhu)和三口采油井(S12-4、S12-5和S12-19)的油藏微生物多样性。基于T-RFLP图谱的多样性指数表明注入水样品具有更丰富的细菌和古菌多样性。相似性指数表明,样品间细菌群落结构的相似性介于22.4%～30.8%之间,古菌群落结构的相似性介于20.8%～34.5%之间。查询RDP数据库推测这4个油藏样品所共有的优势微生物可能为Pseudomonas属,Marinobacter属和产甲烷微生物。T-RFLP技术能方便快捷的分析微生物多样性,其在油藏微生物多样性研究上的应用可以为MEOR提供有用的信息。     

复合垂直流人工湿地系统中不同植物根际微生物群落结构

为了研究复合垂直流人工湿地在夏季和秋季运行期间湿地中不同植物根际微生物群落的结构特征,采用PCR-DGGE技术和基因测序技术,对复合垂直流人工湿地系统中梭鱼草(Pontederia cordata)、美人蕉(Canna indica)、再力花(Thalia dealbata)3种植物根际微生物群落的结构特征和组成多样性进行分析。结果表明,3种植物根际微生物的种类和数量各不相同。人工湿地下行池中梭鱼草、美人蕉、再力花根际微生物的Shannon指数分别为2.91、2.96和2.90,说明美人蕉根际能更好地富集周围的微生物,提高微生物群落的多样性。通过比较各种植物根际微生物的相似度,发现根际微生物群落结构存在一定的季节差异;美人蕉和再力花根际微生物群落结构相似度较高(65%以上)。微生物群落组成多样性的结果显示,复合垂直流人工湿地系统中3种植物在实验期间根际微生物群落分属8个纲:α-变形菌纲(14.37%)、β-变形菌纲(38.13%)、γ-变形菌纲(6.87%)、ε-变形菌纲(16.87%)、δ-变形菌纲(6.87%)、放线菌纲(8.76%)、拟杆菌纲(4.38%)、硝化螺旋菌纲(3.75%)。β-变形菌纲为该人工湿地系统试验期间不同植物根际富集的主要细菌类群。本实验为以后人工湿地系统植物优化选择提供了一定的理论依据。     

峨眉山与黄山藏酋猴肠道菌群组成的比较

峨眉山和黄山都是我国著名的藏酋猴Macaca thibetana生态旅游地,对两地藏酋猴肠道微生物群落结构的比较研究,有助于了解不同生境、不同旅游管理方式对野生灵长类动物的影响。本研究对峨眉山藏酋猴M. t.thibetana肠道微生物16S rRNA基因进行测序,并与黄山藏酋猴M. t. huangshanensis肠道微生物的群落结构进行了比较。结果显示,两地藏酋猴肠道菌群有大量共有的可操作分类单元,而群落组成和多样性方面存在较大差异。峨眉山藏酋猴肠道菌群的优势门类为厚壁菌门Firmicutes(69. 04%±11. 81%)、拟杆菌门Bacteroidetes(21. 59%±10. 05%)和放线菌门Actinobacteria (2. 73%±2. 17%);黄山藏酋猴为厚壁菌门(46. 34%±8. 15%)、拟杆菌门(36. 75%±6. 38%)和变形菌门Proteobacteria(14. 91%±8. 06%)。在属级水平上,峨眉山藏酋猴肠道丰度最高的为颤螺菌属Oscillospira(23. 49%±16. 63%),黄山藏酋猴为普氏菌属Prevotella(36. 35%±9. 15%)。在群落多样性方面,黄山藏酋猴α多样性指数显著低于峨眉山,且两者的菌群结构也产生了显著分化。PICRUSt功能富集分析显示,峨眉山藏酋猴在脂类代谢、外源化学物的降解与代谢等通路显著富集,而黄山藏酋猴在多糖的合成与代谢等通路显著富集。研究还发现峨眉山藏酋猴肠道存在一定丰度的传染性致病菌,这可能与峨眉山的生态旅游有关。     

油藏微生物多样性的分子生态学研究进展

油藏微生物是一类宝贵的资源, 在油层生态系统的物质循环和能量流动中发挥着主导作用。传统上, 主要依赖纯培养技术, 使得大部分油藏微生物都没能得到充分认识。分子方法克服了纯培养方法中遇到的问题, 能更好地了解环境微生物群落多样性。近年来, 在油藏微生物群落多样性研究中的应用进展迅速, 对油藏微生物学和生态学的发展产生了重要影响。文中评述了近年来国内外在油藏微生物分子生态方面的研究进展, 并对进一步的研究提出了展望。     

油藏微生物多样性的分子生态学研究进展

油藏微生物是一类宝贵的资源,在油层生态系统的物质循环和能量流动中发着主导作用.传统上,主要依赖纯培养技术,使得大部分油藏微生物都没能得到充分认识.分子方法克服了纯培养方法中遇到的问题,能更好地了解环境微生物群落多样性.近年来,在油藏微生物群落多样性研究中的应用进展迅速,对油藏微生物学和生态学的发展产生了重要影响.文中评述了近年来国内外在油藏微生物分子生态方面的研究进展,并对进一步的研究提出了展望.     

油藏微生物及其压力适应机制

目前我国油田开发主要处于高含水后期,微生物驱提高石油采收率技术(MEOR)以低成本、环境友好等独特的优势引起了石油工业界的重视。实际上,经过半个多世纪的发展,MEOR已经成为提高采收率的重要前沿技术。高压是油藏的主要环境特征,在影响油藏微生物生存与活性等方面具有重要作用。本文从油藏及其微生物的主要特征、微生物对高压环境的适应机制以及高压下微生物降解烃的代谢特征等方面进行了综述。介绍了对油藏微生物资源、群落结构、微生物在油水相中分布的认识,微生物乳化原油机制,以及微生物在油藏厌氧环境中协同代谢、受温度和压力影响的特点,并列举了MEOR的矿场应用。在高压适应机制上,微生物主要通过改变和调整细胞膜结构、增加胞内脂质组分和表达胞内特殊酶等作用来实现对压力的适应;在高压下烃降解微生物代谢速率低于常压,而且耐压菌和嗜压菌具有不同的烃降解效率。     

Molecular analysis of the bacterial community in a continental high-temperature and water-flooded petroleum reservoir

Water from a continental high-temperature, long-term water-flooded petroleum reservoir in Huabei Oilfield in China was analysed for its bacterial community and diversity. The bacteria were characterized by their 16S rRNA genes. A 16S rRNA gene clone library was constructed from the community DNA, and using restriction fragment length polymorphism analysis, 337 randomly selected clones were clustered with 74 operational taxonomic units. Sequencing and phylogenetic analyses showed that the screened clones were affiliated with Gammaproteobacteria (85.7%), Thermotogales (6.8%), Epsilonproteobacteria (2.4%), low-G+C Gram-positive (2.1%), high-G+C Gram-positive, Betaproteobacteria and Nitrospira (each <1.0%). Thermopilic bacteria were found in the high-temperature water from the flooded petroleum reservoir, as well as mesophilic bacteria such as Pseudomonas-like clones. The mesophilic bacteria were probably introduced into the reservoir as it was being exploited. This work provides significant information on the structure of bacterial communities in high-temperature, long-term water-flooded petroleum reservoirs.     

Molecular phylogenetic diversity of the microbial community associated with a high-temperature petroleum reservoir at an offshore oilfield

The microbial community and its diversity in production water from a high-temperature, water-flooded petroleum reservoir of an offshore oilfield in China were characterized by 16S rRNA gene sequence analysis. The bacterial and archaeal 16S rRNA gene clone libraries were constructed from the community DNA and, using sequence analysis, 388 bacterial and 220 archaeal randomly selected clones were clustered with 60 and 28 phylotypes, respectively. The results showed that the 16S rRNA genes of bacterial clones belonged to the divisions Firmicutes, Thermotogae, Nitrospirae and Proteobacteria, whereas the archaeal library was dominated by methanogen-like rRNA genes (Methanothermobacter, Methanobacter, Methanobrevibacter and Methanococcus), with a lower percentage of clones belonging to Thermoprotei. Thermophilic microorganisms were found in the production water, as well as mesophilic microorganisms such as Pseudomonas and Acinetobacter-like clones. The thermophilic microorganisms may be common inhabitants of geothermally heated specialized subsurface environments, which have been isolated previously from a number of high-temperature petroleum reservoirs worldwide. The mesophilic microorganisms were probably introduced into the reservoir as it was being exploited. The results of this work provide further insight into the composition of microbial communities of high-temperature petroleum reservoirs at offshore oilfields.     

胜利油田采油区土壤石油污染状况及其微生物群落结构

基于不同开采年代新油井(2011—)和老油井(1966—2003年)周边土壤的调查取样,研究了采油区土壤石油污染状况,利用PCR-DGGE和克隆测序技术,探讨了新、老油井周边土壤微生物的群落结构.结果表明:油井周边土壤均受到不同程度的石油污染,其石油烃含量大多高于土壤石油污染临界值(500 mg·kg^-1),且老油井周边土壤污染水平更高.污染土壤石油烃含量与土壤有机碳、全氮和速效钾含量呈显著正相关.老油井周边土壤微生物群落多样性指数随污染水平的增大而减小,新油井则呈相反的趋势.DGGE图谱优势条带测序结果表明,油井周边土壤均存在明显的优势菌,大多为石油烃相关菌和烃类降解菌,如微杆菌属、链霉菌属、迪茨氏菌属、黄杆菌属及α、γ变形菌等.
     

Microbial diversity in degraded and non-degraded petroleum samples and comparison across oil reservoirs at local and global scales

Microorganisms have shown their ability to colonize extreme environments including deep subsurface petroleum reservoirs. Physicochemical parameters may vary greatly among petroleum reservoirs worldwide and so do the microbial communities inhabiting these different environments. The present work aimed at the characterization of the microbiota in biodegraded and non-degraded petroleum samples from three Brazilian reservoirs and the comparison of microbial community diversity across oil reservoirs at local and global scales using 16S rRNA clone libraries. The analysis of 620 16S rRNA bacterial and archaeal sequences obtained from Brazilian oil samples revealed 42 bacterial OTUs and 21 archaeal OTUs. The bacterial community from the degraded oil was more diverse than the non-degraded samples. Non-degraded oil samples were overwhelmingly dominated by gammaproteobacterial sequences with a predominance of the genera Marinobacter and Marinobacterium. Comparisons of microbial diversity among oil reservoirs worldwide suggested an apparent correlation of prokaryotic communities with reservoir temperature and depth and no influence of geographic distance among reservoirs. The detailed analysis of the phylogenetic diversity across reservoirs allowed us to define a core microbiome encompassing three bacterial classes (Gammaproteobacteria, Clostridia, and Bacteroidia) and one archaeal class (Methanomicrobia) ubiquitous in petroleum reservoirs and presumably owning the abilities to sustain life in these environments.

     

不同压力条件下油藏内源细菌群落激活过程中变性梯度凝胶电泳分析?

摘要：【目的】了解常压（1 MPa）和高压（10 MPa）条件下内源细菌激活中的细菌群落和结构的变化。【方法】利用胜利油田沾3×24井产出液水样,在常压和高压下进行富集培养后,定时取样,样品用变性梯度凝胶电泳（denature gradient gel electrophoresis,DGGE）技术分析。【结果】通过对内源微生物激活前后DGGE条带的数量和亮度变化分析表明,油藏环境中的细菌在种类和数量上并不丰富,激活剂的加入改变了菌群原有的贫营养环境,从而使一些因营养缺乏生长受抑制细菌得以大量繁殖,菌群结     

Characterisation of culture-independent and -dependent microbial communities in a high-temperature offshore chalk petroleum reservoir

Recent studies have indicated that oil reservoirs harbour diverse microbial communities. Culture-dependent and culture-independent methods were used to evaluate the microbial diversity in produced water samples of the Ekofisk oil field, a high temperature, and fractured chalk reservoir in the North Sea. DGGE analyses of 16S rRNA gene fragments were used to assess the microbial diversity of both archaeal and bacterial communities in produced water samples and enrichment cultures from 4 different wells (B-08, X-08, X-18 and X-25). Low diversity communities were found when 16S rDNA libraries of bacterial and archaeal assemblages were generated from total community DNA obtained from produced water samples and enrichment cultures. Sequence analysis of the clones indicated close matches to microbes associated with high-temperature oil reservoirs or other similar environments. Sequences were found to be similar to members of the genera Thermotoga, Caminicella, Thermoanaerobacter, Archaeoglobus, Thermococcus, and Methanobulbus. Enrichment cultures obtained from the produced water samples were dominated by sheathed rods. Sequence analyses of the cultures indicated predominance of the genera Petrotoga, Arcobacter, Archaeoglobus and Thermococcus. The communities of both produced water and enrichment cultures appeared to be dominated by thermophilic fermenters capable of reducing sulphur compounds. These results suggest that the biochemical processes in the Ekofisk chalk reservoir are similar to those observed in high-temperature sandstone reservoirs.