渤海N油田油藏内源微生物群落结构分析

利用变性梯度凝胶电泳(denaturing gradient gel electrophoresis,DGGE)技术分析渤海N油田注水和未注水油藏中细菌和古菌群落结构组成及分布特征,试分析注水过程对油藏微生物群落丰度和种群的影响,为开展目标油田本源微生物采油试验提供技术支持。结果显示,注水采油井中细菌丰度和种类明显高于未注水采油井,其中注水采油井中的细菌主要为固氮螺菌属(Azospira sp.)、铜绿假单胞菌(Pseudomonas sp.)、陶厄氏菌属(Thauera sp.);注水井古菌的丰度和种类与未注水井也存在一定差异,主要为热自养甲烷热杆菌(Methanothermobacter thermautotrophicus)、甲烷鬃毛菌属(Methanosaeta)、嗜泉古菌(Crenarchaeote)。注水井和未注水井中的细菌、古菌种类分布有限,但丰度较高,主要为提高原油采收率有益的采油菌种,显示该区块具备开展本源微生物微生物采油技术实施的条件。     

DGGE技术监测生物制氢反应器微生物群落结构和演替

为了研究生物制氢反应器微生物群落结构, 揭示混合菌群的生态学效能. 从连续流生物制氢反应器CSTR运行不同时期取活性污泥, 利用变性梯度凝胶电泳(DGGE)技术研究了产氢混合菌群的多样性和动态变化. 研究表明, 反应器从启动到乙醇型发酵稳定的运行, 经历了明显的微生物群落结构演替过程, 28天后反应器微生物群落结构基本恒定, 形成顶级群落. 16S rDNA 序列同源性分析表明, 优势种群为低G + C含量革兰氏阳性细菌分支的Clostridium sp.和Ethanologenbacterium sp., b变形菌亚纲的Acidovorax sp., g变形菌亚纲的Kluyvera sp.和一些未被培养的拟杆菌群的细菌和螺旋体. 21天后产氢细菌Ethanologenbacterium sp., Clostridium sp. (Clostridiaceae bacterium 80 Kb)和一些未被培养的螺旋体群细菌的数量明显增加, 形成乙醇型发酵群落, 产氢量大幅度提高. 群落经过演替微生物多样性增强后降低, 在群落演替过程中一直存在的Clostridium sp., sp., Kluyvera sp.和未被培养的拟杆菌群等是构成群落结构的基本种群, 混合菌群之间存在着共代谢作用, 共同决定产氢效能.     

黄海北部不同站位海洋细菌群落分布特征

[目的]为揭示北黄海不同海域中真细菌群落分布的差异,[方法]采用16s rRNA基因文库和变性梯度凝胶电泳(DGGE)技术,对远海和近海两个站位的沉积物和水体中细菌群落特征进行了解析和评价.[结果]文库分析揭示海水及沉积物中微生物种类丰富,存在大量未被认知的类群.各站位中主要为变形菌门(Proteobacteria),沉积物中γ-Proteobacteria和δ-Proteobacteria亚门占优势,水中则以α-Proteobacteria亚门占优势,但各亚门微生物在两个站位中存在明显系统发育学分歧.DGGE图谱聚类分析显示,近海沉积物和海水中细菌群落优势类群相似性很高,而远海沉积物和海水中则相似性很低.[结论]研究结果表明,微生物种类在不同地理位置和生存介质中存在明显差异,环境因素对微生物的分布起主导作用.     

青藏高原淡水湖普莫雍错和盐水湖阿翁错湖底沉积物中细菌群落的垂直分布

【目的】湖泊沉积物中存储着大量独特的微生物,这些微生物在湖泊生态系统生物地球化学循环中扮演着非常重要的角色。然而,很少有研究报道微生物群落在湖泊沉积物中的垂直分布。本文比较研究青藏高原淡水湖普莫雍错和盐水湖阿翁错沉积物在不同深度下细菌的丰度和群落结构。【方法】利用定量PCR(q PCR)和变性梯度凝胶电泳(DGGE)技术分别测定细菌群落的丰度与群落结构。【结果】定量PCR结果显示,湖泊沉积物中细菌丰度均随深度增加而降低,盐水湖阿翁错和淡水湖普莫雍错的细菌丰度分别从1011数量级降到108数量级,从1012数量级降到1010数量级。在相对应的沉积物层,淡水湖沉积物的细菌丰度比盐水湖高1-2个数量级。变性梯度凝胶电泳(DGGE)指纹图谱的分析表明,淡水湖沉积物细菌群落的DGGE条带数(丰富度)显著高于盐水湖(P=0.014);淡水与盐水湖泊沉积物细菌群落结构明显不同,同时在同一湖泊沉积物中上层(0-6 cm)和下层(7-20 cm)细菌群落结构也呈明显分异。系统发育分析表明,盐水湖阿翁错沉积物特有菌门为Gamma-变形菌、拟杆菌门、蓝细菌和栖热菌门,而淡水湖普莫雍错沉积物中特有菌门为Delta-和Beta-变形菌、酸杆菌和绿弯菌门。【结论】青藏高原淡水与盐水湖泊沉积物细菌丰度与群落结构具有明显的差异;同时,细菌群落结构在沉积物的不同深度也表现出差异。这些结果可为进一步阐明青藏高原湖泊生态系统中微生物对气候环境变化的响应提供科学依据。     

3种淡水环境中低核酸含量细菌的滤过性

【目的】增加低核酸含量(LNA)细菌与过滤性细菌之间的认识。【方法】采用流式细胞技术(FCM)、变性梯度凝胶电泳(DGGE)及统计学分析研究3种典型淡水环境中细菌群落与滤过性。【结果】LNA细菌与过滤性细菌在数量上具有很好的相关性(y=0.646x+22.42,R2=0.984,P0.01),细菌的滤过性不仅与细菌大小有关,还与细胞整体形状及其伸缩性有关;细菌群落组织与LNA细菌比重呈负相关性,而与HNA细菌呈正相关性。【结论】0.45μm膜过滤对水样微生物群落中的LNA细菌具有极强的筛选效果;细菌群落组织与其基于流式细胞技术测定的基因含量具有密切联系。     

凉水保护区土壤产类漆酶-多铜氧化酶细菌群落结构

在凉水国家级自然保护区3种主要林型红松(Pinus koraiensis)、白桦(Betula platyphylla)及云杉(Picea dietrich)林采集林下土壤样品,以铜离子作为筛选剂处理后,结合平板分离法与基于16S rDNA V3区片段的变性梯度凝胶电泳(DenaturingGradient Gel Electrophoresis,DGGE)技术,调查了土壤样品中产类漆酶-多铜氧化酶(laccase-like multicopper oxidase,LMCO)细菌的群落结构。这是研究产类漆酶-多铜氧化酶细菌在环境中存在的种、属及分布的新尝试。平板分离获得10株细菌均为芽孢杆菌属(Bacillus sp.),其中梭状芽孢杆菌(Bacillus fusiformis)未见相关报道。通过DGGE图谱分析可知,产类漆酶-多铜氧化酶细菌在研究地不同林型土壤中的群落结构无明显差异,在红松林土壤中多样性最为丰富。DGGE条带测序结果表明,取样地土壤中产类漆酶细菌主要为罗尔斯顿菌属(Ralstonia sp.)、肠杆菌属(Enterobacter sp.)、芽孢杆菌属和一些未培养细菌。     

钉螺体内细菌群落结构PCR-DGGE分析方法的建立

建立了通过PCR-DGGE研究钉螺体内细菌群落结构的方法,并采用此技术分析了钉螺体内细菌群落结构.钉螺样本采自湖北省荆州市,样本带回实验室经破壳解剖后取清洗的钉螺内脏放入无菌离心管后经酚-氯仿法抽提微生物DNA.使用获得的微生物DNA为模板,选择细菌原核通用引物F357-GC和R518进行PCR扩增得到大约250 bp的目的片段,然后采用变性梯度凝胶电泳结合DNA测序技术对获得的目的片段进行分析.结果显示此技术能够区分幼螺和成螺体内细菌群落结构差异,并发现在成螺体内有大量的Pseudomonas属和Acidobacteria属细菌出现.     

PCR-DGGE方法分析原油储层微生物群落结构及种群多样性

使用基于 16 S r DNA的 PCR- DGGE(变性梯度凝胶电泳 )图谱分析结合条带割胶回收 DNA进行序列分析 ,对新疆克拉玛依油田一中区注水井 (12 # 9- 11)和与该注水井相应的两个采油井 (12 # 9- 9S、13# 11- 8)井口样品微生物群落的多样性进行了比较并鉴定了部分群落成员。 DGGE图谱聚类分析表明注水井与两油井微生物群落的相似性分别为 30 %和 2 0 % ,而两油井间微生物群落结构的相似性为 5 4 %。DGGE图谱中优势条带序列分析表明注水井样品和油井样品中的优势菌群为未培养的环境微生物 ,它们与数据库中 α、γ、δ、ε变形杆菌 (Proteobacteria)和拟杆菌 (Bacteroidetes)有很近的亲缘关系。 DGGE与分子克隆相结合的分子生物学方法在研究微生物提高原油采收率 (MEOR)机理 ,以及指导 MEOR在油田生产中的应用有着重要的意义     

大庆油田某油层石油微生物区系分布

早在1926年苏联与美国学者均在汕出油层水中发现了不同种类微生物群系。此后,油田微生物区系的研究引起了人们的广泛重视。许多学者在油田的岩心和原油中以及油层水中,发现了大量的微生物群系。如脱氮细菌、硫酸盐还原菌、甲烷生成菌、石油分解菌和石腊分解     

太湖浮游细菌与春末浮游藻类群落结构演替的相关分析

为研究浮游细菌与浮游藻类群落演替的相关性,2005年4月至6月在太湖5个观测点采集浮游细菌及浮游藻类样本。分别采用聚合酶链式反应-变性梯度凝胶电泳（PCR—DGGE）和显微观察的方法分析浮游细菌及浮游藻类群落组成。结果表明,春末夏初,浮游细菌与藻类均呈现较高的多样性,浮游细菌DGGE图谱具有43种不同条带,浮游藻类的常见种有29种。浮游细菌群落聚类分析显示,丝藻（Ulothrix sp．）和微囊藻（Microcystis spp．）占优势时,浮游细菌群落基因组成存在明显差异。以藻类种群Shannon—Wiener多样性指数（Hp）,浮游藻类总细胞数（N）以及Microcystis spp．（M）百分含量为变量,典型对应分析（CCA）结果显示浮游细菌与浮游藻类群落结构变化的相关系数为30．9％,表明春末夏初太湖浮游细菌与浮游藻类群落演替具有较高的相关性。     

Bacterial communities in a crude oil gathering and transferring system (China)

Bacterial communities in crude oil and oil field production water samples from an oil gathering and transferring system in Changqing Oil field in China were investigated by 16S rRNA denaturing gradient gel electrophoresis (DGGE) analysis followed by gene cloning and sequencing. DGGE profiles showed that bacterial communities are far more rich in the water samples than that in the crude oil samples, and that bacteria related to Ochrobactrum sp. and Stenotrophomonas sp. were detected in all crude oil and oil field water samples. Bacteria related to Burkholderia sp., Brevundimonas sp., and Propionibacterium sp. were detected in the crude oil samples but not in water samples. Bacteria related to Hippea sp., Acidovorax sp., Arcobacter sp., Pseudomonas sp., Thiomicrospira sp., Brevibacterium sp., Tissierella sp. and Peptostreptococcus sp. were detected in the water samples but not in crude oil samples. Using an archaea-specific primer set, methanogens related to Methanomicrobials and Methanosarcinales were found in water samples but not in crude oil samples. The comparability of the microbial communities in the water and crude oil phase during the period of oil gathering and transferring process was 83.3% and 88.2%, respectively, indicating a stable structure of the microbial communities.     

胜利油藏不同时间细菌群落结构的比较

利用聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)和构建16S rRNA基因克隆文库2种方法,对孤岛油田两口井(注水井G和采油井L)在相距9个月的2个时间点(A和B)所采集样品的细菌群落结构进行了比较。DGGE图谱聚类分析表明注水井在2个时间点的微生物群落结构相似性为48.1%,而采油井的相似性只有28.7%。16S rRNA基因克隆文库结果表明,A时间点样品G中的优势菌群为Betaproteobacteria、Gammaproteobacteria,还有Deferribacteres、Firmicutes、Bacteroidetes等;而样品L中,Gammaproteobacteria中的Moraxellaceae含量达到97%。B时间点G中除了优势菌Betaproteobacteria之外,Deferribacteres的数量显著增加,成为优势菌;而L在B时间点优势菌除Gammaproteobacteria外,还有Betaproteobacteria和Firmicutes。采油井中的微生物群落结构随时间发生了显著改变,而注水井变化不显著。这一结果部分揭示了微生物采油过程中地层微生物群落的变化规律,有助于进一步阐明微生物驱油的机理。     

培养法和免培养法联合检测油藏环境烃降解菌和产甲烷菌群多样性

烃降解菌和产甲烷菌是油藏环境微生物生态系统中重要的功能菌群, 采用DGGE和FISH方法分析了不同油藏样品中两类菌群的多样性和产甲烷活性。DGGE结果表明, 不同水样的alkB基因多样性相差较大, 而且注水井条带明显多于采油井。FISH结果表明, 油藏水样中产甲烷菌含量明显高于烃降解菌, 且两者空间分布的位置较近; 说明油藏环境中烃降解菌和产甲烷菌结成一定的相互关系。富集培养表明, 胜利油田产出液接种物培养130 d后, 石油烃降解率达到50%以上, 产甲烷的最大速率达到1.57×10^?2 mmol/(L?d)。利用分子生物学方法分析油藏环境功能菌群的多样性, 可以为开展微生物采油技术的应用提供有用信息。     

Phylogenetic diversity of bacterial communities associated with bioremediation of crude oil in microcosms

Bioremediation, mainly by indigenous bacteria, has been regarded as an effective way to clean up oil pollution after an oil spill. In order to obtain a systematic understanding of the succession of bacterial communities associated with oil bioremediation, sediments collected from the Penglai 19-3 oil platform were co-incubated with crude oil. Oil biodegradation was assessed on the basis of changes in oil composition monitored by GC–MS. Changes in the bacterial community structure were detected by two 16S rRNA gene based culture-independent methods, denaturing gradient gel electrophoresis (DGGE) and clone library. The results suggested that crude oil was rapidly degraded during the 30-day bioremediation period. Bacteria affiliated with the genus Pseudomonas dominated all three clone libraries. But dramatic changes were also detected in the process of biodegradation of crude oil. The “professional hydrocarbonocastic bacteria” (e.g., Alcanivorax) became abundant in the two samples during the bioremediation period. Meanwhile, δ-proteobacteria was only detected in the two samples. Information on the bacterial community revealed in this study will be useful in developing strategies for bioremediation of crude oil dispersed in the marine ecosystem.     

Firmicutes is an Important Component of Microbial Communities in Water-Injected and Pristine Oil Reservoirs,Western Siberia,Russia

The dominant microbial components of fluids from wells in pristine and water-injected, high-temperature, Western Siberian oil fields, were analyzed by PCR-DGGE. Particular emphasis was placed on sulphate-reducing organisms, due to their ecological and industrial importance. Bacterial phylotypes obtained from the non-water-injected Stolbovoye oil field were more diverse than those from the Samotlor field, which is subject to secondary oil recovery by reinjection of recycled production water. The majority of phylotypes from both sites were related to Firmicutes. The low similarity to their closest relatives indicates unique bacterial communities in deep underground production waters and crude oil. Archaeal phylotypes detected only in the Samotlor samples were represented by Methanosarcinales and Methanobacteriales.     

Monitoring exogenous and indigenous bacteria by PCR-DGGE technology during the process of microbial enhanced oil recovery

A field experiment was performed to monitor changes in exogenous bacteria and to investigate the diversity of indigenous bacteria during a field trial of microbial enhanced oil recovery (MEOR). Two wells (26-195 and 27-221) were injected with three exogenous strains and then closed to allow for microbial growth and metabolism. After a waiting period, the pumps were restarted and the samples were collected. The bacterial populations of these samples were analyzed by denaturing gradient gel electrophoresis (DGGE) with PCR-amplified 16S rRNA fragments. DGGE profiles indicated that the exogenous strains were retrieved in the production water samples and indigenous strains could also be detected. After the pumps were restarted, average oil yield increased to 1.58 and 4.52 tons per day in wells 26-195 and 27-221, respectively, compared with almost no oil output before the injection of exogenous bacteria. Exogenous bacteria and indigenous bacteria contributed together to the increased oil output. Sequence analysis of the DGGE bands revealed that Proteobacteria were a major component of the predominant bacteria in both wells. Changes in the bacteria population in the reservoirs during MEOR process were monitored by molecular analysis of the 16S rRNA gene sequence. DGGE analysis was a successful approach to investigate the changes in microorganisms used for enhancing oil recovery. The feasibility of MEOR technology in the petroleum industry was also demonstrated.     

Influence of crude oil on changes of bacterial communities in Arctic sea-ice

The danger of a petroleum hydrocarbon spillage in the polar, ice-covered regions is increasing due to oil exploration in Arctic offshore areas and a growing interest in using the Northern Sea Route (NSR) as an alternative transportation route for Arctic oil and gas. However, little is known about the potential impact of accidental oil spills on this environment. We investigated the impact of crude oil on microbial community composition in six different Arctic sea-ice samples incubated with crude oil at 1 degrees C in microcosms for one year. Alterations in the composition of bacterial communities were analyzed with the culture-independent molecular methods DGGE (denaturing gradient gel electrophoresis) and FISH (fluorescence in situ hybridization). DGGE, FISH and cultivation methods revealed a strong shift in community composition toward the gamma-proteobacteria in sea-ice and melt pool samples incubated with crude oil. Marinobacter spp., Shewanella spp. and Pseudomonas spp. were the predominant phylotypes in the oil-treated microcosms. The ability of indigenous sea-ice bacteria to degrade hydrocarbons at low temperature (1 degrees C) was tested using four representative strains cultivated from sea-ice enriched with crude oil. [14C]Hexadecane was degraded by the sea-ice isolates at 20-50% capacity of the mesophilic type strain Marinobacter hydrocarbonoclasticus, a known hydrocarbon degrader, incubated at 22 degrees C.     

Monitoring of microbial community structure and succession in the biohydrogen production reactor by denaturing gradient gel electrophoresis (DGGE)

Biohydrogen production has been concerned ex-tremely as a new technology of energy resource pro-duction by many scientists[1—4]. Enhancement of hy-drogen production efficiency and cutting down the operating cost are very important problems, which are the limiting factors for the industrialization of hydro-gen production process. The fermentation hydrogen production technology offers a new method to resolve these difficulties[5—8]. Compared with photosynthetic hydrogen production possesses, f…     

Monitoring of microbial community structure and succession in the biohydrogen production reactor by denaturing gradient gel electrophoresis (DGGE)

To study the structure of microbial communities in the biological hydrogen production reactor and determine the ecological function of hydrogen producing bacteria, anaerobic sludge was obtained from the continuous stirred tank reactor (CSTR) in different periods of time, and the diversity and dynamics of microbial communities were investigated by denaturing gradient gel electrophoresis (DGGE). The results of DGGE demonstrated that an obvious shift of microbial population happened from the beginning of star-up to the 28th day, and the ethanol type fermentation was established. After 28 days the structure of microbial community became stable, and the climax community was formed. Comparative analysis of 16S rDNA sequences from reamplifying and sequencing the prominent bands indicated that the dominant population belonged to low G+C Gram-positive bacteria (Clostridium sp. andEthanologenbacterium sp.), β-proteobacteria (Acidovorax sp.), γ-proteobacteria (Kluyvera sp.), Bacteroides (uncultured bacterium SJA-168), and Spirochaetes (uncultured eubacterium E1-K13), respectively. The hydrogen production rate increased obviously with the increase ofEthanologenbacterium sp.,Clostridium sp. and uncultured Spirochaetes after 21 days, meanwhile the succession of ethanol type fermentation was formed. Throughout the succession the microbial diversity increased however it decreased after 21 days. Some types ofClostridium sp.Acidovorax sp.,Kluyvera sp., and Bacteroides were dominant populations during all periods of time. These special populations were essential for the construction of climax community. Hydrogen production efficiency was dependent on both hydrogen producing bacteria and other populations. It implied that the cometabolism of microbial community played a great role of biohydrogen production in the reactors.     

Changes in Microbial Communities,Including both Uncultured and Culturable Bacteria,with Mid-Ocean Ballast-Water Exchange during a Voyage from Japan to Australia

We assessed changes in the microbial communities in ballast water during a trans-Pacific voyage from Japan to Australia that included a mid-ocean ballast-water exchange. Uncultured (i.e., total) and culturable bacteria were counted and were characterized by using denaturing gradient gel electrophoresis (DGGE). There was a clear decrease over time in numbers of uncultured microorganisms, except for heterotrophic nanoflagellates, whereas the abundance of culturable bacteria initially decreased after the ballast-water exchange but then increased. The increase, however, was only up to 5.34% of the total number of uncultured bacteria. Cluster analysis showed that the DGGE profiles of uncultured bacteria clearly changed after the exchange. In contrast, there was no clear change in the DGGE profiles of culturable bacteria after the exchange. Multidimensional scaling analysis showed changes in microbial communities over the course of the voyage. Although indicator microbes as defined by the International Convention for the Control and Management of Ships'' Ballast Water and Sediments were occasionally detected, no coliform bacteria were detected after the exchange.