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。     

Nitrification and Autotrophic Nitrifying Bacteria in a Hydrocarbon-Polluted Soil

In vitro ammonia-oxidizing bacteria are capable of oxidizing hydrocarbons incompletely. This transformation is accompanied by competitive inhibition of ammonia monooxygenase, the first key enzyme in nitrification. The effect of hydrocarbon pollution on soil nitrification was examined in situ. In a microcosm study, adding diesel fuel hydrocarbon to an uncontaminated soil (agricultural unfertilized soil) treated with ammonium sulfate dramatically reduced the amount of KCl-extractable nitrate but stimulated ammonium consumption. In a soil with long history of pollution that was treated with ammonium sulfate, 90% of the ammonium was transformed into nitrate after 3 weeks of incubation. Nitrate production was twofold higher in the contaminated soil than in the agricultural soil to which hydrocarbon was not added. To assess if ammonia-oxidizing bacteria acquired resistance to inhibition by hydrocarbon, the contaminated soil was reexposed to diesel fuel. Ammonium consumption was not affected, but nitrate production was 30% lower than nitrate production in the absence of hydrocarbon. The apparent reduction in nitrification resulted from immobilization of ammonium by hydrocarbon-stimulated microbial activity. These results indicated that the hydrocarbon inhibited nitrification in the noncontaminated soil (agricultural soil) and that ammonia-oxidizing bacteria in the polluted soil acquired resistance to inhibition by the hydrocarbon, possibly by increasing the affinity of nitrifying bacteria for ammonium in the soil.     

Nitrification and autotrophic nitrifying bacteria in a hydrocarbon-polluted soil.

In vitro ammonia-oxidizing bacteria are capable of oxidizing hydrocarbons incompletely. This transformation is accompanied by competitive inhibition of ammonia monooxygenase, the first key enzyme in nitrification. The effect of hydrocarbon pollution on soil nitrification was examined in situ. In a microcosm study, adding diesel fuel hydrocarbon to an uncontaminated soil (agricultural unfertilized soil) treated with ammonium sulfate dramatically reduced the amount of KCl-extractable nitrate but stimulated ammonium consumption. In a soil with long history of pollution that was treated with ammonium sulfate, 90% of the ammonium was transformed into nitrate after 3 weeks of incubation. Nitrate production was twofold higher in the contaminated soil than in the agricultural soil to which hydrocarbon was not added. To assess if ammonia-oxidizing bacteria acquired resistance to inhibition by hydrocarbon, the contaminated soil was reexposed to diesel fuel. Ammonium consumption was not affected, but nitrate production was 30% lower than nitrate production in the absence of hydrocarbon. The apparent reduction in nitrification resulted from immobilization of ammonium by hydrocarbon-stimulated microbial activity. These results indicated that the hydrocarbon inhibited nitrification in the noncontaminated soil (agricultural soil) and that ammonia-oxidizing bacteria in the polluted soil acquired resistance to inhibition by the hydrocarbon, possibly by increasing the affinity of nitrifying bacteria for ammonium in the soil.     

Selective substrate utilization by marine hydrocarbonoclastic bacteria

Several strains of bacteria, isolated from marine environments, were characterized for their hydrocarbon oxidizing abilities using a complex synthetic mixture of hydrocarbons. Attempts were made at a broad classification of these organisms on the basis of their behavior towards four major groups of hydrocarbons, normal paraffins, iso-paraffins, cyclo-paraffins, and aromatics, known to be present in crude oils. Although bacteria appear to be able to oxidize hydrocarbons at random, this study has shown that it may be possible to recognize a rudimental pattern if we view their oxidative abilities in terms of groups of hydrocarbons rather than individual compounds. A study of the action of combined strains on the synthetic hydrocarbon mixture was performed. It was found that no particular benefit could be derived as compared to the use of single strains.     

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。     

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。     

OLAND生物脱氮系统中硝化菌群16S rDNA的DGGE分析

为了考察生物脱氮系统中硝化菌群（氨氧化菌和亚硝酸氧化菌）的种群多样性及硝化菌群随溶解氧降低的种群变化规律,并建立一套行之有效的用于自养生物脱氮系统中功能微生物菌群的快速分子检测技术,采用DGGE（变性梯度凝胶电泳）分子检测技术对硝化菌群的16SrDNA的特异性PCR扩增产物进行了分析,结果表明：OLAND生物脱氮系统中氨氧化菌和亚硝酸氧化菌随溶解氧的降低表现出了不同的种群变化规律,氨氧化菌种群多样性受溶解氧的影响非常大,而非亚硝酸氧化菌的种群多样性比较单一,且不受溶解氧的影响。结合FISH（全细胞荧光原位杂交）分析结果表明,在OLAND限氧稳定运行后期,亚硝化单胞菌属（Nitrosomonas）是主要的氨氧化菌,占OLAND限氧亚硝化阶段反应器中总细菌数的72.5%左右。     

Ladderane phospholipids in anammox bacteria comprise phosphocholine and phosphoethanolamine headgroups

Anammox bacteria present in wastewater treatment systems and marine environments are capable of anaerobically oxidizing ammonium to dinitrogen gas. This anammox metabolism takes place in the anammoxosome which membrane is composed of lipids with peculiar staircase-like 'ladderane' hydrocarbon chains that comprise three or four linearly concatenated cyclobutane structures. Here, we applied high-performance liquid chromatography coupled to electrospray ionization tandem mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to elucidate the full identity of these ladderane lipids. This revealed a wide variety of ladderane lipid species with either a phosphocholine or phosphoethanolamine polar headgroup attached to the glycerol backbone. In addition, in silico analysis of genome data gained insight into the machinery for the biosynthesis of the phosphocholine and phosphoethanolamine phospholipids in anammox bacteria.     

Evidence of Signatures for Recognition of Hydrocarbon Microseepage in El Hajeb Oilfield,Eastern Tunisia

Microbial prospecting technique was applied in El Hajeb oilfield, Tunisia for potential hydrocarbon resource exploration by examining the anomalous abundance of hydrocarbon oxidizing bacteria. The method depends on the seepage of light gaseous hydrocarbons such as C1 – C5 from the petroleum pools to the shallow surface giving the reasonable conditions to the development of highly specialized bacterial population. A total of 51 soil samples were collected from 1.5 m depth. Bacterial counts for n-propane utilizing bacteria ranged between nil and 2.44?×?10⁵?cfu/gm. The light gaseous hydrocarbons analysis showed that their concentrations were 102, 55, 41, 20, 21, 19, 10 and 141 ppb for C1, C2, C3, iC4, nC4, iC5, nC5 and ΣC2⁺, respectively. The gas concentration maps showed the presence of dynamic hydrocarbons micro-seepage from petroleum systems. The integrated geomicrobial and adsorbed soil gas results demonstrated that the anomalous hydrocarbon zones spread in all territory which could presumably help to evaluate highpotentialareas. In conclusion, our study confirms that the use of microbial data and adsorbed gas analyses of soil samples is a low cost tool for hydrocarbon exploration. This technique has been confirmed and can be applied successfully in frontier basins.     

油气微生物勘探机理及应用

【目的】微生物油气勘探技术是基于油气藏的轻烃微渗漏原理衍生的地表勘探技术。油气藏中的轻烃部分(C1-C5)以微渗漏的方式通过上覆的沉积层,在近地表土壤中诱导专门利用轻烃的微生物繁殖与生长,油气区的微生物种类与浓度有别于下伏没有油气藏的地区。通过分析微生物的浓度异常特征,对油气富集区及油气藏进行研究和预测。【方法】在人工模拟条件下研究油气微生物数量和群落异常特征,在此基础上,以海相碳酸盐岩气田普光气田为研究对象,进一步开展微生物勘探研究,鉴定油气藏上方气态烃氧化过程的微生物驱动者,提取土壤中的微生物异常信息。【结果】人工模拟条件下发现Lacibacter cauensis、Methylococcaceae、Methylophilaceae与甲烷气体培养正相关(气指示菌),而未培养的硫氧化微生物等则与丁烷培养正相关(油指示菌)。【结论】进一步在普光气田原位研究中进行验证,发现地表油气微生物数量和群落异常与油气藏有较好的关联性;与油气化探指标对比后发现,油气藏上方微生物正异常和轻烃负异常具有较为明显的互补关系。本研究深化了对典型油气藏上方气态烃氧化微生物转化机制的认识,为油气微生物勘探技术提供理论与实践依据。     

Analysis of stable low molecular weight (LMW) RNA profiles of hydrocarbon metabolizing bacteria by staircase electrophoresis

Staircase electrophoresis (SCE) in polyacrilamide gels was used to analyze the stable low-molecular weight (LMW) RNA profiles of several propane and butane oxidizing bacteria belonging to different species and genera. Differences in the number and distribution of the RNA bands in these profiles allowed us to differentiate among them. Congruent results were found between the established classification of these bacteria and results obtained by LMW RNA profiling and moreover, some misclassified strains can be assigned to the correct genus and species using this technique. LMW RNA profiling by staircase electrophoresis, which makes possible the analysis of a large number of strains in a short time, permits rapid identification of hydrocarbon metabolizing species when compared with LMW RNA profiles of reference strains.     

人工湿地黑臭水体处理系统微生物脱氮机理研究

以上海市老段浦I、II和北夏3座水平潜流人工湿地黑臭河道处理系统为研究对象,进行了水平潜流湿地处理黑臭河道氨氮的转化及脱氮机理的研究。研究表明,3座人工湿地的pH值均呈弱碱性,且沿湿地水流方向变化较小。溶解氧值在0.09—0.35mg/L范围内波动,氨氮沿湿地的流向呈递减的趋势,亚硝态氮及硝态氮浓度较低。在老段浦人工湿地的同一土样中,亚硝化细菌的数量远大于硝化细菌的数量,北夏人工湿地中,湿地前端的亚硝化细菌与硝化细菌的数量近似相等,但在湿地末端亚硝化细菌数量要远小于硝化细菌的数量。原位曝气抑制反硝化反应试验研究表明,3座人工湿地都发生了"新"的脱氮途径-短程硝化-反硝化反应,其中两座老段浦人工湿地50%的氮以短程硝化-反硝化反应去除。北夏人工湿地中约20%的氮以短程硝化反硝化的途径去除。       

Filamentous sulfur bacteria preserved in modern and ancient phosphatic sediments: implications for the role of oxygen and bacteria in phosphogenesis

Marine phosphate‐rich sedimentary deposits (phosphorites) are important geological reservoirs for the biologically essential nutrient phosphorous. Phosphorites first appear in abundance approximately 600 million years ago, but their proliferation at that time is poorly understood. Recent marine phosphorites spatially correlate with the habitats of vacuolated sulfide‐oxidizing bacteria that store polyphosphates under oxic conditions to be utilized under sulfidic conditions. Hydrolysis of the stored polyphosphate results in the rapid precipitation of the phosphate‐rich mineral apatite—providing a mechanism to explain the association between modern phosphorites and these bacteria. Whether sulfur bacteria were important to the formation of ancient phosphorites has been unresolved. Here, we present the remains of modern sulfide‐oxidizing bacteria that are partially encrusted in apatite, providing evidence that bacterially mediated phosphogenesis can rapidly permineralize sulfide‐oxidizing bacteria and perhaps other types of organic remains. We also describe filamentous microfossils that resemble modern sulfide‐oxidizing bacteria from two major phosphogenic episodes in the geologic record. These microfossils contain sulfur‐rich inclusions that may represent relict sulfur globules, a diagnostic feature of modern sulfide‐oxidizing bacteria. These findings suggest that sulfur bacteria, which are known to mediate the precipitation of apatite in modern sediments, were also present in certain phosphogenic settings for at least the last 600 million years. If polyphosphate‐utilizing sulfide‐oxidizing bacteria also played a role in the formation of ancient phosphorites, their requirements for oxygen, or oxygen‐requiring metabolites such as nitrate, might explain the temporal correlation between the first appearance of globally distributed marine phosphorites and increasing oxygenation of Neoproterozoic oceans.     

Microbial assimilation of hydrocarbons. I. The fine-structure of a hydrocarbon oxidizing Acinetobacter sp.

1. The fine-structure analysis of the hydrocarbon oxidizing microorganism, Acinetobacter sp., demonstrated a cytoplasmic modification resulting from growth on paraffinic and olefinic hydrocarbons. 2. Intracytoplasmic hydrocarbon inclusions were documented by electron microscopy with chemical identifications obtained by gas chromatography and X-ray diffraction. 3. These results demonstrate the ability of a microorganism to accumulate hydrocarbon substrates intracellularly which, in turn, indicates the transport across the cell membrane.     

Biomineralisation of manganese on titanium surfaces exposed to seawater

A 2-year long study was carried out to isolate and characterise various bacterial species present in the biofilm formed on titanium surfaces exposed to seawater and to assess the manganese oxidizing potential of the marine isolates. The amount of manganese present in the biofilm was also measured using atomic absorption spectrometry (AAS). The results showed that titanium was susceptible to biofouling. More than 50% of the culturable marine bacterial isolates were capable of bringing about oxidation of Mn(II). All these manganese oxidizing bacteria were heterotrophic. Autotrophic manganese oxidizing bacteria such as Leptothrix was not isolated in the present study. The AAS results confirmed that the manganese content in the biofilms increased with increasing exposure time. Hence, the study indicates that the titanium surfaces when exposed to seawater were colonised by a large number of heterotrophic bacteria, which have the ability of bringing about biomineralisation of manganese.     

Abundance of archaeal and bacterial ammonia oxidizers – Possible bioindicator for soil monitoring

Many soil functions are driven by soil microorganisms and they have therefore been identified as appropriate indicators for monitoring of soil status. Genetic profiling of the bacterial ammonia oxidizing community was recently top-scored as soil biological indicator (Ritz et al., 2009). However, ammonia oxidation is not only performed by bacteria, but also ammonia oxidizing archaea. Based on the suggested niche differentiation between these two groups and findings that they are susceptible to environmental change in soil ecosystems at varying scales, we suggest that the abundance of these two communities rather than community profiling of the ammonia oxidizing bacteria could serve as a relevant and cost-effective bioindicator for soil monitoring.     

太湖沉积物中氮循环菌的微生态

采用荧光原位杂交(FISH)技术,对梅梁湾和贡湖湾湖区沉积物中微生物的主要种群,及氮循环功能微生物的数量和分布进行了研究,发现随着深度增加,细菌和古菌数量均逐渐减少,但古菌在总菌数中所占比例有所增加;梅梁湾沉积物中氨氧化细菌及亚硝酸氧化细菌的数量均高于贡湖湾,随深度增加,两者数量均逐渐减少,但在贡湖湾其占总菌数的比例高于梅梁湾,表明水生植物可能对沉积物中微生物组成及氮循环有重要影响。泉古菌在太湖沉积物中普遍存在且数量高于氨氧化细菌,说明其在淡水湖泊中对氮循环可能有重要作用。     

Pure bacterial isolates that convert p-xylene to terephthalic acid

Bacteria that grow on p-xylene, p-toluic acid, and terephthalic acid (TPA) were isolated from a wastewater bioreactor that is used to treat a waste stream that contains all three of these compounds. Although previously described aerobic bacteria degrade p-xylene by initially oxidizing a single methyl group to form p-toluic acid and then cleaving the aromatic ring, some of the bacteria isolated during this study transformed p-xylene by oxidizing both methyl groups to produce TPA.     

Barite encrustation of benthic sulfur‐oxidizing bacteria at a marine cold seep

Crusts and chimneys composed of authigenic barite are found at methane seeps and hydrothermal vents that expel fluids rich in barium. Microbial processes have not previously been associated with barite precipitation in marine cold seep settings. Here, we report on the precipitation of barite on filaments of sulfide‐oxidizing bacteria at a brine seep in the Gulf of Mexico. Barite‐mineralized bacterial filaments in the interiors of authigenic barite crusts resemble filamentous sulfide‐oxidizing bacteria of the genus Beggiatoa. Clone library and iTag amplicon sequencing of the 16S rRNA gene show that the barite crusts that host these filaments also preserve DNA of Candidatus Maribeggiatoa, as well as sulfate‐reducing bacteria. Isotopic analyses show that the sulfur and oxygen isotope compositions of barite have lower δ³⁴S and δ¹⁸O values than many other marine barite crusts, which is consistent with barite precipitation in an environment in which sulfide oxidation was occurring. Laboratory experiments employing isolates of sulfide‐oxidizing bacteria from Gulf of Mexico seep sediments showed that under low sulfate conditions, such as those encountered in brine fluids, sulfate generated by sulfide‐oxidizing bacteria fosters rapid barite precipitation localized on cell biomass, leading to the encrustation of bacteria in a manner reminiscent of our observations of barite‐mineralized Beggiatoa in the Gulf of Mexico. The precipitation of barite directly on filaments of sulfide‐oxidizing bacteria, and not on other benthic substrates, suggests that sulfide oxidation plays a role in barite formation at certain marine brine seeps where sulfide is oxidized to sulfate in contact with barium‐rich fluids, either prior to, or during, the mixing of those fluids with sulfate‐containing seawater in the vicinity of the sediment/water interface. As with many other geochemical interfaces that foster mineral precipitation, both biological and abiological processes likely contribute to the precipitation of barite at marine brine seeps such as the one studied here.     

Acetylene Reduction Assays for Nitrogen Fixation Freshwaters: a Note of Caution

Lake water samples were observed to transform [14-C]ethylene into water-soluble compounds that were undetectable by conventional acetylene reduction assay procedures. Methane oxidizing bacteria, which are known to be common in freshwaters, appeared to be responsible for this activity. As much as 28 percent of added ethylene has been observed to be transformed and this figure is probably an underestimate. It is suggested that acetylene reduction assays may not be accurately applied to samples containing methane oxidizing bacteria.