胜利油田单12高温油藏非培养和富集培养样品的细菌组成特性

[目的]通过比较分析油藏样品的微生物群落结构特点,认识油藏微生物的生态功能.[方法]利用3种油藏微生物研究中常用的富集培养方法,对胜利油田单12区块S12-4油井产出水样品进行了选择性富集培养,运用构建16S rRNA基因文库的方法分析了富集样品和非培养样品的细菌多样性.[结果]通过16S rRNA基因序列比对发现,非培养样品、异养菌富集样品、烃降解菌富集样品和硫酸盐还原菌富集样品中的优势菌分别为Pseudomonas属,Thermotoga属,Thermaerobacter属和Thermotoga属的成员.多样性分析结果表明,非培养样品的微生物多样性最丰富,同时非培养样品和富集样品的微生物群落结构存在很大的差异,富集样品中的微生物包括优势菌在油藏原位环境中含量很低.[结论]细菌组成差异的比较结果,对油藏微生物的生态功能研究和微生物驱油潜力评估具有重要意义.     

Bacterial, archaeal and eukaryotic diversity of smooth and pustular microbial mat communities in the hypersaline lagoon of Shark Bay

The bacterial, archaeal and eukaryotic populations of nonlithifying mats with pustular and smooth morphology from Hamelin Pool, Shark Bay were characterised using small subunit rRNA gene analysis and microbial isolation. A highly diverse bacterial population was detected for each mat, with 16S rDNA clones related to Actinobacteria, Bacteroidetes, Chloroflexi, Cyanobacteria, Gemmatimonas, Planctomycetes, Alphaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Verrucomicrobia and candidate division TM6 present in each mat. Spirochaetes were detected in the smooth mat only, whereas candidate division OP11 was only detected in the pustular mat. Targeting populations with specific primers revealed additional cyanobacterial diversity. The archaeal population of the pustular mat was comprised purely of Halobacteriales, whereas the smooth mat contained 16S rDNA clones from the Halobacteriales, two groups of Euryarchaea with no close characterised matches, and the Thaumarchaea. Nematodes and fungi were present in each mat type, with diatom 18S rDNA clones only obtained from the smooth mat, and tardigrade and microalgae clones only retrieved from the pustular mat. Cultured isolates belonged to the Firmicutes, Gammaproteobacteria, Alphaproteobacteria, Bacteroidetes, Actinobacteria, Cyanobacteria, and Halobacteriales. The mat populations were significantly more diverse than those previously reported for Hamelin Pool stromatolites, suggesting specific microbial populations may be associated with the nonlithifying and lithifying microbial communities of Hamelin Pool.     

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.     

Massive dominance of Epsilonproteobacteria in formation waters from a Canadian oil sands reservoir containing severely biodegraded oil

The subsurface microbiology of an Athabasca oil sands reservoir in western Canada containing severely biodegraded oil was investigated by combining 16S rRNA gene- and polar lipid-based analyses of reservoir formation water with geochemical analyses of the crude oil and formation water. Biomass was filtered from formation water, DNA was extracted using two different methods, and 16S rRNA gene fragments were amplified with several different primer pairs prior to cloning and sequencing or community fingerprinting by denaturing gradient gel electrophoresis (DGGE). Similar results were obtained irrespective of the DNA extraction method or primers used. Archaeal libraries were dominated by Methanomicrobiales (410 of 414 total sequences formed a dominant phylotype affiliated with a Methanoregula sp.), consistent with the proposed dominant role of CO(2) -reducing methanogens in crude oil biodegradation. In two bacterial 16S rRNA clone libraries generated with different primer pairs, >?99% and 100% of the sequences were affiliated with Epsilonproteobacteria (n?=?382 and 72 total clones respectively). This massive dominance of Epsilonproteobacteria sequences was again obtained in a third library (99% of sequences; n?=?96 clones) using a third universal bacterial primer pair (inosine-341f and 1492r). Sequencing of bands from DGGE profiles and intact polar lipid analyses were in accordance with the bacterial clone library results. Epsilonproteobacterial OTUs were affiliated with Sulfuricurvum, Arcobacter and Sulfurospirillum spp. detected in other oil field habitats. The dominant organism revealed by the bacterial libraries (87% of all sequences) is a close relative of Sulfuricurvum kujiense - an organism capable of oxidizing reduced sulfur compounds in crude oil. Geochemical analysis of organic extracts from bitumen at different reservoir depths down to the oil water transition zone of these oil sands indicated active biodegradation of dibenzothiophenes, and stable sulfur isotope ratios for elemental sulfur and sulfate in formation waters were indicative of anaerobic oxidation of sulfur compounds. Microbial desulfurization of crude oil may be an important metabolism for Epsilonproteobacteria indigenous to oil reservoirs with elevated sulfur content and may explain their prevalence in formation waters from highly biodegraded petroleum systems.     

A molecular phylogenetic survey of sea-ice microbial communities (SIMCO)

16S rDNA clone library analysis was used to identify bacterial biodiversity in a variety of sea-ice microbial communities (SIMCO). DNA was extracted from seven Antarctic sea-ice samples and one Arctic sea-ice sample and 16S rDNA PCR-amplified using universal and Archaea-specific primers. Recombinant 16S rDNA clones were obtained and dereplicated using restriction fragment length polymorphism analysis (RFLP). After RFLP analysis, 100 distinct phylotypes (a unique clone or group of clones with sequence similarity of >0.98) were defined. From the clone libraries 16S rDNA sequences of bacterial and eukaryotic origin were detected, however Archaea were not detected either with universal or Archaea-specific 16S rDNA primer sets. Bacterial phylotypes grouped within the alpha and gamma proteobacteria, the Cytophaga-Flavobacterium-Bacteroides division, the Gram-Positive bacteria and the orders Chlamydiales and Verrucomicrobiales. The majority of bacterial phylotypes were affiliated with heterotrophic taxa and many grouped closely with cultivated genera and species. Eukaryotic clones were affiliated with a variety of autotrophic and heterotrophic nanoplankton and included a large number of chloroplast 16S rDNA genes. The findings of this investigation corroborated culture data indicating bacterial biodiversity increased in SIMCO displaying high levels of primary production, however the bacterial communities within SIMCO were highly heterogeneous at the genus/species-level between different samples. A comparison of Antarctic and Arctic SIMCO revealed certain sea-ice dwelling bacterial genera are common at both poles.     

Bacterial community structure and diversity in a century-old manure-treated agroecosystem

Changes in soil microbial community structure and diversity may reflect environmental impact. We examined 16S rRNA gene fingerprints of bacterial communities in six agroecosystems by PCR amplification and denaturing gradient gel electrophoresis (PCR-DGGE) separation. These soils were treated with manure for over a century or different fertilizers for over 70 years. Bacterial community structure and diversity were affected by soil management practices, as evidenced by changes in the PCR-DGGE banding patterns. Bacterial community structure in the manure-treated soil was more closely related to the structure in the untreated soil than that in soils treated with inorganic fertilizers. Lime treatment had little effect on bacterial community structure. Soils treated with P and N-P had bacterial community structures more closely related to each other than to those of soils given other treatments. Among the soils tested, a significantly higher number of bacterial ribotypes and a more even distribution of the bacterial community existed in the manure-treated soil. Of the 99 clones obtained from the soil treated with manure for over a century, two (both Pseudomonas spp.) exhibited 100% similarity to sequences in the GenBank database. Two of the clones were possible chimeras. Based on similarity matching, the remaining 97 clones formed six major clusters. Fifty-six out of 97 were assigned taxonomic units which grouped into five major taxa: alpha-, beta-, and gamma-Proteobacteria (36 clones), Acidobacteria (16 clones), Bacteroidetes (2 clones), Nitrospirae (1 clone), and Firmicutes (1 clone). Forty-one clones remained unclassified. Results from this study suggested that bacterial community structure was closely related to agroecosystem management practices conducted for over 70 years.     

High-Density Universal 16S rRNA Microarray Analysis Reveals Broader Diversity than Typical Clone Library When Sampling the Environment

Molecular approaches aimed at detection of a broad-range of prokaryotes in the environment routinely rely on classifying heterogeneous 16S rRNA genes amplified by polymerase chain reaction (PCR) using primers with broad specificity. The general method of sampling and categorizing DNA has been to clone then sequence the PCR products. However, the number of clones required to adequately catalog the majority of taxa in a sample is unwieldy. Alternatively, hybridizing target sequences to a universal 16S rRNA gene microarray may provide a more rapid and comprehensive view of prokaryotic community composition. This study investigated the breadth and accuracy of a microarray in detecting diverse 16S rRNA gene sequence types compared to clone-and-sequencing using three environmental samples: urban aerosol, subsurface soil, and subsurface water. PCR products generated from universal 16S rRNA gene-targeted primers were classified by using either the clone-and-sequence method or by hybridization to a novel high-density microarray of 297,851 probes complementary to 842 prokaryotic subfamilies. The three clone libraries comprised 1391 high-quality sequences. Approximately 8% of the clones could not be placed into a known subfamily and were considered novel. The microarray results confirmed the majority of clone-detected subfamilies and additionally demonstrated greater amplicon diversity extending into phyla not observed by the cloning method. Sequences matching operational taxonomic units within the phyla Nitrospira, Planctomycetes, and TM7, which were uniquely detected by the array, were verified with specific primers and subsequent amplicon sequencing. Subfamily richness detected by the array corresponded well with nonparametric richness predictions extrapolated from clone libraries except in the water community where clone-based richness predictions were greatly exceeded. It was concluded that although the microarray is unreliable in identifying novel prokaryotic taxa, it reveals greater diversity in environmental samples than sequencing a typically sized clone library. Furthermore, the microarray allowed samples to be rapidly evaluated with replication, a significant advantage in studies of microbial ecology.     

A novel archaeal group in the phylum Crenarchaeota found unexpectedly in an eukaryotic survey in the Cariaco Basin

Archaea have been found in many more diverse habitats than previously believed due in part to modern molecular approaches to discovering microbial diversity. We report here an unexpected expansion of the habitat diversity of the Archaea in the Cariaco Basin we found using a primer set designed for 18S eukaryotic rDNA sequence analysis. The results presented here expand the originally identified 9 archaeal clones reported in this environment using bacterial/archaeal primers to 152 archaeal clones: 67 (18 OTU) of these clones were found at a depth of 900 m of station A while 71 (9 OTU) of them were at a depth of between 300 approximately 335 m of station B&C depending upon which location the samples were taken. We used three phylogenetic analysis methods and detected 20 phylotypes belonging to a single previously unreported group distantly related to the Crenarchaeota. Also, we determined that the original nine sequences did not fall into any of the known phyla of the Archaea suggesting that they may represent a novel group within the Kingdom Archaea. Thus, from these two studies, we suggest that Archaea in the Cariaco Basin could be unique; however, further studies using archaeal-specific primers and the design of new primers as well as the systematic use of several different primer combinations may improve the chances of understanding the archeal diversity in the Cariaco Basin.     

Methanogenesis,sulfate reduction and crude oil biodegradation in hot Alaskan oilfields

Petrochemical and geological evidence suggest that petroleum in most reservoirs is anaerobically biodegraded to some extent. However, the conditions for this metabolism and the cultivation of the requisite microorganisms are rarely established. Here, we report on microbial hydrocarbon metabolism in two distinct oilfields on the North Slope of Alaska (designated Fields A and B). Signature anaerobic hydrocarbon metabolites were detected in produced water from the two oilfields offering evidence of in situ biodegradation activity. Rate measurements revealed that sulfate reduction was an important electron accepting process in Field A (6–807 µmol S l^?1 day^?1), but of lesser consequence in Field B (0.1–10 µmol S l^?1 day^?1). Correspondingly, enrichments established at 55°C with a variety of hydrocarbon mixtures showed relatively high sulfate consumption but low methane production in Field A incubations, whereas the opposite was true of the Field B enrichments. Repeated transfer of a Field B enrichment showed ongoing methane production in the presence of crude oil that correlated with ≥ 50% depletion of several component hydrocarbons. Molecular‐based microbial community analysis of the methanogenic oil‐utilizing consortium revealed five bacterial taxa affiliating with the orders Thermotogales, Synergistales, Deferribacterales (two taxa) and Thermoanaerobacterales that have known fermentative or syntrophic capability and one methanogen that is most closely affiliated with uncultured clones in the H₂‐using family Methanobacteriaceae. The findings demonstrate that oilfield‐associated microbial assemblages can metabolize crude oil under the thermophilic and anaerobic conditions prevalent in many petroleum reservoirs.     

Comprehensive Analysis of Secondary Dental Root Canal Infections: A Combination of Culture and Culture-Independent Approaches Reveals New Insights

Persistence of microorganisms or reinfections are the main reasons for failure of root canal therapy. Very few studies to date have included culture-independent methods to assess the microbiota, including non-cultivable microorganisms. The aim of this study was to combine culture methods with culture-independent cloning methods to analyze the microbial flora of root-filled teeth with periradicular lesions. Twenty-one samples from previously root-filled teeth were collected from patients with periradicular lesions. Microorganisms were cultivated, isolated and biochemically identified. In addition, ribosomal DNA of bacteria, fungi and archaea derived from the same samples was amplified and the PCR products were used to construct clone libraries. DNA of selected clones was sequenced and microbial species were identified, comparing the sequences with public databases. Microorganisms were found in 12 samples with culture-dependent and -independent methods combined. The number of bacterial species ranged from 1 to 12 in one sample. The majority of the 26 taxa belonged to the phylum Firmicutes (14 taxa), followed by Actinobacteria, Proteobacteria and Bacteroidetes. One sample was positive for fungi, and archaea could not be detected. The results obtained with both methods differed. The cloning technique detected several as-yet-uncultivated taxa. Using a combination of both methods 13 taxa were detected that had not been found in root-filled teeth so far. Enterococcus faecalis was only detected in two samples using culture methods. Combining the culture-dependent and –independent approaches revealed new candidate endodontic pathogens and a high diversity of the microbial flora in root-filled teeth with periradicular lesions. Both methods yielded differing results, emphasizing the benefit of combined methods for the detection of the actual microbial diversity in apical periodontitis.     

The phylogeography of Adelie penguin faecal flora

The gut of animals changes quickly from a totally sterile environment before birth to a numerous and highly diverse microbial community shortly after birth. However, few studies have examined the source of the bacteria colonizing the gut. We used a genetic based approach to investigate the distribution and acquisition of faecal microbial communities by Adelie penguins, Pygoscelis adeliae , breeding in the Ross Sea region of Antarctica by cloning a portion of the 16S rRNA gene and by automated ribosomal intergenic spacer analysis (ARISA). We hypothesized that bacteria were either acquired from the penguins' neighbours or inherited from their ancestors. Samples were collected from Adelie penguin breeding colonies at the north-western edge of the Ross Sea coast through to the southernmost Adelie penguin colonies on Ross Island. Most of the bacterial sequences we obtained were only distantly homologous with previously published sequences. Bacterial taxa appear to have a restricted distribution as the majority of 16S rRNA clones were isolated from only one or two hosts. Faecal bacterial community similarity was strongly negatively correlated with the genetic distance between hosts suggesting that bacterial communities are inherited. There was little support for a correlation between distance between collection sites and community similarity.     

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.     

新疆泥火山细菌遗传多样性

为了解新疆乌苏泥火山细菌多样性,从泥火山泥浆样品中直接提取总DNA,构建了含150个有效转化子的泥火山细菌16S rDNA基因文库,转化子经菌液PCR及HaeⅢ酶切后获得16个不同带型,克隆测序结果表明,其分属于16个不同的分类单元.一部分序列与已知细菌类群的16S rDNA序列相似性较高,归属变形菌门(Proteobacteria),厚壁菌门(Firmicutes),梭杆菌门(Fusobacteria),放线菌门(Actinobacteria);另外一部分序列与已知细菌类群的16S rDNA序列同源性较低,可能代表新的分类单位.研究结果显示,泥火山环境中微生物种群丰富,值得进一步研究.     

Characterization of microbial diversity and community in water flooding oil reservoirs in China

The diversity and distribution of bacterial and archaeal communities in four different water flooding oil reservoirs with different geological properties were investigated using 16S rDNA clone library construction method. Canonical correspondence analysis was used to analyze microbial community clustering and the correlation with environmental factors. The results indicated that the diversity and abundance in the bacterial communities were significantly higher than the archaeal communities, while both of them had high similarity within the communities respectively. Phylogenetic analysis showed that of compositions of bacterial communities were distinctly different both at phylum and genus level. Proteobacteria dominated in each bacterial community, ranging from 61.35 to 75.83?%, in which α-proteobacteria and γ-proteobacteria were the main groups. In comparison to bacterial communities, the compositions of archaeal communities were similar at phylum level, while varied at genus level, and the dominant population was Methanomicrobia, ranging from 65.91 to 92.74?% in the single oil reservoir. The factor that most significantly influenced the microbial communities in these reservoirs was found to be temperature. Other environmental factors also influenced the microbial communities but not significantly. It is therefore assumed that microbial communities are formed by an accumulated effect of several factors. These results are essential for understanding ecological environment of the water flooding oil reservoirs and providing scientific guidance to the performance of MEOR technology.     

Bacterial diversity in biofilms formed on condenser tube surfaces in a nuclear power plant

To elucidate the bacterial diversity in biofilms formed on a condenser tube from a nuclear power plant, 16S rRNA gene sequences were examined using a PCR-cloning-sequencing approach. Twelve operational taxonomic units were retrieved in the clone library, and the estimated species richness was low (13.2). Most of the clones (94.7%) were affiliated with α-Proteobacteria; Planctomycetes and γ-Proteobacteria were much rarer. Interestingly, except for one clone belonging to Pseudoalteromonas, most of the sequences displayed sequence similarities <97% of those of the closest type strains. Based on 16S rRNA phylogenetic analysis, most bacteria were assigned to novel taxa above the species level. The low species richness and unusual bacterial composition may be attributable to selective pressure from chlorine in the cooling water. To prevent or control bacterial biofilms in cooling circuits, additional studies of the physiology and ecology of these species will be essential.     

Microbial community analysis of a coastal hot spring in Kagoshima, Japan, using molecular- and culture-based approaches

Ibusuki hot spring is located on the coastline of Kagoshima Bay, Japan. The hot spring water is characterized by high salinity, high temperature, and neutral pH. The hot spring is covered by the sea during high tide, which leads to severe fluctuations in several environmental variables. A combination of molecular- and culture-based techniques was used to determine the bacterial and archaeal diversity of the hot spring. A total of 48 thermophilic bacterial strains were isolated from two sites (Site 1: 55.6°C; Site 2: 83.1°C) and they were categorized into six groups based on their 16S rRNA gene sequence similarity. Two groups (including 32 isolates) demonstrated low sequence similarity with published species, suggesting that they might represent novel taxa. The 148 clones from the Site 1 bacterial library included 76 operational taxonomy units (OTUs; 97% threshold), while 132 clones from the Site 2 bacterial library included 31 OTUs. Proteobacteria, Bacteroidetes, and Firmicutes were frequently detected in both clone libraries. The clones were related to thermophilic, mesophilic and psychrophilic bacteria. Approximately half of the sequences in bacterial clone libraries shared <92% sequence similarity with their closest sequences in a public database, suggesting that the Ibusuki hot spring may harbor a unique and novel bacterial community. By contrast, 77 clones from the Site 2 archaeal library contained only three OTUs, most of which were affiliated with Thaumarchaeota.     

Characterization of the prokaryotic diversity through a stratigraphic permafrost core profile from the Qinghai-Tibet Plateau

Permafrost on the Qinghai-Tibet Plateau is one of the most sensitive regions to climate warming, thus characterizing its microbial diversity and community composition may be important for understanding their potential responses to climate changes. Here, we investigated the prokaryotic diversity in a 10-m-long permafrost core from the Qinghai-Tibet Plateau by restriction fragment length polymorphism analysis targeting the 16S rRNA gene. We detected 191 and 17 bacterial and archaeal phylotypes representing 14 and 2 distinct phyla, respectively. Proteobacteria was the dominant bacterial phylum, while archaeal communities were characterized by a preponderance of Thaumarchaeota. Some of prokaryotic phylotypes were closely related to characterized species involved in carbon and nitrogen cycles, including nitrogen fixation, methane oxidation and nitrification. However, the majority of the phylotypes were only distantly related to known taxa at order or species level, suggesting the potential of novel diversity. Additionally, both bacterial α diversity and community composition changed significantly with sampling depth, where these communities mainly distributed according to core horizons. Arthrobacter-related phylotypes presented at high relative abundance in two active layer soils, while the deeper permafrost soils were dominated by Psychrobacter-related clones. Changes in bacterial community composition were correlated with most measured soil variables, such as carbon and nitrogen contents, pH, and conductivity.